Compositions and methods for treating non-age-associated hearing impairment in a human subject

ABSTRACT

Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No.PCT/US2021/018919, filed Feb. 19, 2021, which claims priority to U.S.Provisional Patent Application Ser. No. 62/979,792, filed Feb. 21, 2020,the entire contents of which are herein incorporated by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing XML which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Oct. 5, 2022, isname 4833_0050002_Seqlisting_ST26.xml and is 970,899 bytes in size.

TECHNICAL FIELD

The present disclosure relates generally to the use of nucleic acids totreat hearing loss in a human subject.

BACKGROUND OF THE DISCLOSURE

The ear is a complex organ, classically described as including the outerear, the middle ear, the inner ear, the hearing (acoustic) nerve and theauditory system (which processes sound as it travels from the ear to thebrain). In addition to detecting sound, the ear also helps to maintainbalance. Thus, disorders of the inner ear can cause hearing loss,tinnitus, vertigo and imbalance. Vertigo is a hallucination of motion,and is the cardinal symptom of vestibular system disease. Vertigo can becaused by problems in the inner ear or central nervous system. Commoninner ear causes of vertigo include: vestibular neuritis (sudden,unilateral vestibular loss), Meniere's disease (episodic vertigo),benign paroxysmal positional vertigo (BPPV), and bilateral vestibularloss. Common central nervous system causes of vertigo include:post-concussion syndrome, cervical vertigo, vestibular migraine,cerebrovascular disease, and acoustic neuroma.

Hearing loss is one of the most common human sensory deficits, and canoccur for many reasons. Some people may be born with hearing loss whileothers may lose their hearing slowly over time. Presbycusis (alsospelled presbyacusis) is age-related hearing loss. Approximately 36million American adults report some degree of hearing loss, and one inthree people older than 60 and half of those older than 85 experiencehearing loss.

Hearing loss can be the result of environmental factors or a combinationof genetic and environmental factors. About half of all people who havetinnitus—phantom noises in their auditory system (ringing, buzzing,chirping, humming, or beating—also have an over-sensitivity to/reducedtolerance for certain sound frequency and volume ranges, known ashyperacusis (also spelled hyperacousis). Williams syndrome (also knownas Williams-Beuren Syndrome) is a multisystem disorder caused by thehemizygous deletion of a 1.6 Mb region at 7q11.23 encompassing about 26genes, including the gene encoding LIM kinase 1 (LIMKI). Individualswith Williams Syndrome manifest hyperacusis and progressive hearingloss, and hyperacusis early onset suggests that it could be associatedwith one of the deleted genes.

Environmental causes of hearing loss include certain medications,specific infections before or after birth, and exposure to loud noiseover an extended period. Hearing loss can result from noise, ototoxicagents, presbyacusis, disease, infection or cancers that affect specificparts of the ear. Ischemic damage can cause hearing loss viapathophysiological mechanisms initiated by. As another example,autoimmune inner ear disease (AIED) is characterized by rapidlyprogressive bilateral sensorineural hearing loss, occurring when thebody's immune system attacks cells in the inner ear that are mistakenfor a virus or bacteria.

Approximately 1.5 in 1,000 children are born with profound hearing loss,and another two to three per 1,000 children are born with partialhearing loss (Smith et al., 2005, Lancet 365:879-890). More than half ofthese cases are attributed to a genetic basis (Di Domenico, et al.,2011, J. Cell. Physiol. 226:2494-2499).

Nonsyndromic deafness is hearing loss that is not associated with othersigns and symptoms. In contrast, syndromic deafness involves hearingloss that occurs with abnormalities in other parts of the body. Mostcases of genetic deafness (70 percent to 80 percent) are nonsyndromic;the remaining cases are caused by specific genetic syndromes.

Hearing loss can be conductive (arising from the ear canal or middleear), sensorineural (arising from the inner ear or auditory nerve), ormixed. Most forms of nonsyndromic deafness are associated with permanenthearing loss caused by damage to structures in the inner ear(sensorineural deafness). The great majority of human sensorineuralhearing loss is caused by abnormalities in the hair cells of the organof Corti in the cochlea. There are also very unusual sensorineuralhearing impairments that involve the eighth cranial nerve (thevestibulocochlear nerve) or the auditory portions of the brain. In therarest of these sorts of hearing loss, only the auditory centers of thebrain are affected. In this situation, cortical deafness may occur,where sounds may be heard at normal thresholds, but the quality of thesound perceived is so poor that speech cannot be understood. However,most sensorineural hearing loss is due to poor hair cell function. Thehair cells may be abnormal at birth, or damaged during the lifetime ofan individual. There are both external causes of damage, like noisetrauma and infection, and intrinsic abnormalities, like congenitalmutations to genes that play an important role in cochlear anatomy orphysiology.

Hearing loss that results from changes in the middle ear is calledconductive hearing loss. Some forms of nonsyndromic deafness involvechanges in both the inner ear and the middle ear, called mixed hearingloss. Hearing loss that is present before a child learns to speak isclassified as prelingual or congenital. Hearing loss that occurs afterthe development of speech is classified as postlingual. Most autosomalrecessive loci cause prelingual severe-to-profound hearing loss.

Nonsyndromic deafness can have different patterns of inheritance, andcan occur at any age. Types of nonsyndromic deafness are named accordingto their inheritance patterns. Autosomal dominant forms are designatedDFNA, autosomal recessive forms are DFNB, and X-linked forms are DFN.Each type is also numbered in the order in which it was described. Forexample, DFNA1 was the first described autosomal dominant type ofnonsyndromic deafness.

Between 75 percent and 80 percent of cases are inherited in an autosomalrecessive pattern, which means both copies of the gene in each cell havemutations. Usually, each parent of an individual with autosomalrecessive deafness is a carrier of one copy of the mutated gene, but isnot affected by this form of hearing loss.

Another 20 percent to 25 percent of nonsyndromic deafness cases areautosomal dominant, which means one copy of the altered gene in eachcell is sufficient to result in hearing loss. People with autosomaldominant deafness most often inherit an altered copy of the gene from aparent who has hearing loss.

Between 1 percent and 2 percent of cases show an X-linked pattern ofinheritance, which means the mutated gene responsible for the conditionis located on the X chromosome (one of the two sex chromosomes). Maleswith X-linked nonsyndromic deafness tend to develop more severe hearingloss earlier in life than females who inherit a copy of the same genemutation. A characteristic of X-linked inheritance is that fatherscannot pass X-linked traits to their sons.

Mitochondrial nonsyndromic deafness, which results from changes tomitochondrial DNA, occurs in less than one percent of cases in theUnited States. The altered mitochondrial DNA is passed from a mother toall of her sons and daughters. This type of deafness is not inheritedfrom fathers.

Auditory neuropathy spectrum disorder (ANSD), a hearing disordercharacterized by normal outer hair cells function and abnormal or absentauditory brain stem response, is one of the most common diseases leadingto hearing and speech communication barriers in infants and youngchildren. Approximately 10 percent of children with permanent hearingloss may have ANSD. The OTOF gene is the first gene identified forautosomal recessive non-syndromic ANSD, and mutations in OTOF have beenfound to account for approximately 5% of all cases of autosomalrecessive nonsydromic hearing loss in some populations(Rodriguez-Ballesteros et al. 2008 Human Mut 29(6):823-831).

The causes of nonsyndromic deafness are complex. Researchers haveidentified more than 30 genes that, when altered, are associated withnonsyndromic deafness; however, some of these genes have not been fullycharacterized. Different mutations in the same gene can be associatedwith different types of hearing loss, and some genes are associated withboth syndromic and nonsyndromic deafness.

For example, genes associated with nonsyndromic deafness include, butare not limited to, ATP2B2, ACTG1, CDH23, CLDN14, COCH, COL11A2, DFNA5,DFNB31, DFNB59, ESPN, EYA4, GJB3, KCNQ4, LHFPL5, MYO1A, MYO15A, MYO6,MYO7A, OTOF, PCDH15, SLC26A4, STRC, TECTA, TMC1, TMIE, TMPRSS3, TRIOBP,USHIC, and WFS1.

The most common cause of hearing loss is Nonsyndromic Hearing Loss andDeafness, DFNB1 (also called GJB2-related DFNB1 Nonsyndromic HearingLoss and Deafness; Autosomal Recessive Deafness 1; NeurosensoryNonsyndromic Recessive Deafness 1). Nonsyndromic hearing loss anddeafness (DFNB1) is characterized by congenital, non-progressive,mild-to-profound sensorineural hearing impairment. It is caused bymutations in GJB2 (which encodes the protein connexin 26) and GJB6(which encodes connexin 30). Diagnosis of DFNB1 depends on moleculargenetic testing to identify deafness-causing mutations in GJB2 andupstream cis-regulatory elements that alter the gap junction beta-2protein (connexin 26). Molecular genetic testing of GJB2 detects morethan 99% of deafness-causing mutations in these genes. Unlike some otherforms of hearing loss, DFNB1 nonsyndromic hearing loss and deafness doesnot affect balance or movement. The degree of hearing loss is difficultto predict based on which genetic mutation one has. Even if members ofthe same family are affected by DFNB1 nonsyndromic hearing loss anddeafness, the degree of hearing loss may vary among them.

Mutations in genes coding for connexin26 (Cx26) and/or Cx30 are linkedto approximately half of all cases of human autosomal nonsyndromicprelingual deafness. Cx26 and Cx30 are the two major Cx isoforms foundin the cochlea, and they coassemble to form hybrid (heteromeric andheterotypic) gap junctions (GJs) (Ahmad, et al., Proc. Natl. Acad. Sci.,2007, 104(4):1337-1341). Nonsyndromic hearing loss and deafness, DFNA3,is caused by a dominant-negative pathogenic variant in the GJB2 or GJB6gene, altering either the protein connexin 26 (Cx26) or connexin 30(Cx30), respectively, and is characterized by pre- or postlingual, mildto profound, progressive high-frequency sensorineural hearingimpairment.

OTOF-related deafness (DFNB9 nonsyndromic hearing loss) is characterizedby two phenotypes: prelingual nonsyndromic hearing loss and, lessfrequently, temperature-sensitive nonsyndromic auditory neuropathy(TS-NSAN). Another form of progressive hearing impairment is associatedwith a mutation in the otoferlin gene (e.g., a I1573T mutation or aP1987R mutation, and/or a E1700Q mutation), or is not temperaturesensitive.

Pendred syndrome/DFNB4 (deafness with goiter) is an autosomal recessiveinherited disorder, and accounts for 7.5% of all cases of congenitaldeafness. Pendred syndrome has been linked to mutations in the PDS gene(also known as DFNB4, EVA, PDS, TDH2B and solute carrier family 26,member 4, SLC26A4) on the long arm of chromosome 7 (7931), which encodesthe pendrin protein. Mutations in this gene also cause enlargedvestibular aqueduct syndrome (EVA or EVAS), another congenital cause ofdeafness; specific mutations are more likely to cause EVAS, while othersare more linked with Pendred syndrome. (Azaiez, et al. (December 2007),Hum. Genet. 122 (5): 451-7).

Transmembrane protease, serine 3 is an enzyme encoded by the TMPRSS3gene (also known as DFNB10, DFNB8, ECHOS1, and TADG12). The gene wasidentified by its association with both congenital and childhood onsetautosomal recessive deafness. Mutations in TMPRSS3 are associated withpostlingual and rapidly progressive hearing impairment. The proteinencoded by the TMPRSS3 gene contains a serine protease domain, atransmembrane domain, an LDL receptor-like domain, and a scavengerreceptor cysteine-rich domain. Serine proteases are known to be involvedin a variety of biological processes, whose malfunction often leads tohuman diseases and disorders. This gene is expressed in fetal cochleaand many other tissues, and is thought to be involved in the developmentand maintenance of the inner ear or the contents of the perilymph andendolymph. This gene was also identified as a tumor associated gene thatis overexpressed in ovarian tumors. Four alternatively spliced variantshave been described, two of which encode identical products.

DFN3 deafness is caused by mutations in the POU3F4 gene, which islocated on the X chromosome. In people with this condition, one of thesmall bones in the middle ear (the stapes) cannot move normally, whichinterferes with hearing. This characteristic sign of DFN3 is calledstapes fixation. At least four other regions of the X chromosome areinvolved in hearing loss, but the responsible genes have not beendiscovered. DFNB59 (deafness, autosomal recessive 59), also known asPejvakin or PJVK, is a 352 amino acid protein belonging to the gasderminfamily in vertebrates. DFNB59 is encoded by a gene that maps to humanchromosome 2q31.2, essential for the proper function of auditory pathwayneurons and outer hair cell function. Mutations in DFNB59 are believedto cause non-syndromic sensorineural deafness autosomal recessive type59, a form of sensorineural hearing impairment characterized by absentor severely abnormal auditory brainstem response but normal otoacousticemissions (auditory neuropathy or auditory dys-synchrony). DFNB59 sharessignificant similarity with DFNA5, indicating that these genes share acommon origin.

Alport syndrome is caused by mutations in the COL4A3, COL4A4, and COL4A5genes involved in collagen biosynthesis. Mutations in any of these genesprevent the proper production or assembly of the type IV collagennetwork, which is an important structural component of basementmembranes in the kidney, inner ear, and eye. One of the criteria used indiagnosis of Alport syndrome is bilateral sensorineural hearing loss inthe 2000 to 8000 Hz range. The hearing loss develops gradually, is notpresent in early infancy and commonly presents before the age of 30years.

Defects in ion channels are associated with deafness: DFNA2 nonsyndromichearing loss is inherited as an autosomal dominant mutation in the KCNQ4gene, which encodes the potassium voltage-gated channel subfamily KQTmember 4 also known as voltage-gated potassium channel subunit Kv7.4.DFNA2 nonsyndromic hearing loss is characterized by symmetric,predominantly high-frequency sensorineural hearing loss (SNHL) that isprogressive across all frequencies. At younger ages, hearing loss tendsto be mild in the low frequencies and moderate in the high frequencies;in older persons, the hearing loss is moderate in the low frequenciesand severe to profound in the high frequencies. Although the hearingimpairment is often detected during routine hearing assessment of aschool-age child, it is likely that hearing is impaired from birth,especially at high frequencies. Most affected persons initially requirehearing aids to assist with sound amplification between ages ten and 40years. By age 70 years, all persons with DFNA2 hearing loss havesevere-to-profound hearing impairment.

Mutations in the KCNE1 and KCNQ1 genes cause Jervell and Lange-Nielsensyndrome (JLNS), a type of long QT syndrome, associated with severe,bilateral hearing loss. This condition is an autosomal recessivedisorder that affects an estimated 1.6 to 6 in 1 million children, andis responsible for less than 10 percent of all cases of long QTsyndrome. It has a markedly higher incidence in Norway and Sweden, up to1:200,000. The proteins produced by the KCNE1 and KCNQ1 genes worktogether to form a potassium channel that transports positively chargedpotassium ions out of cells. The movement of potassium ions throughthese channels is critical for maintaining the normal functions of theinner ear and cardiac muscle.

EAST/SeSAME syndrome, characterized by mental retardation, ataxia,seizures, hearing loss, and renal salt waste, is believed to be causedby mutations in KCNJ10 inwardly rectifying potassium channels.

Subjects with Bartter's syndrome with sensorineural deafness type 4(also known as Bartter syndrome IV or BSND) have defects in a Cl-channelaccessory subunit.

Mutations in the ATP6V1B1 gene expressed both in the kidney and in thecochlea are associated with distal renal tubular acidosis (DRTA). Asignificant percentage of children with autosomal recessive DRTA werealso found to experience progressive bilateral sensorineural hearingloss.

Usher syndrome (also known as Hallgren syndrome, Usher-Hallgrensyndrome, retinitis pigmentosa-dysacusis syndrome, and dystrophiaretinae dysacusis syndrome) is a rare disorder caused by a mutation inany one of at least ten genes, resulting in a combination of hearingloss and a gradual visual impairment, and is a leading cause ofdeafblindness. The hearing loss is caused by a defective inner ear,whereas the vision loss results from retinitis pigmentosa (RP), adegeneration of the retinal cells. Usher syndrome has three clinicalsubtypes, denoted as I, II, and III. Subjects with Usher I are bornprofoundly deaf and begin to lose their vision in the first decade oflife, learn to walk slowly as children due to problems in theirvestibular system, and exhibit balance difficulties. Subjects with UsherII are not born deaf, but do have hearing loss, but do not seem to havenoticeable problems with balance; they also begin to lose their visionlater (in the second decade of life) and may preserve some vision eveninto middle age. Subjects with Usher syndrome III are not born deaf, butexperience a gradual loss of their hearing and vision; they may or maynot have balance difficulties.

A mouse model of congenital deafness has been generated by making a nullmutation in the gene encoding the vesicular glutamate transporter-3(VGLUT3). Recently, hearing was restored in the VGLUT3 knockout mouseusing viral-mediated gene therapy (Akil, et al., 2012, Neuron75:283-293).

Math1 (Mouse Homolog of ATH1); also known as HATH1 or Atonal,Drosophila, Homolog of (ATOH1) is essential for hair cell development inthe inner ear; Math1 was therefore proposed to act as a “pro-hair cellgene” in the developing sensory epithelia (Bermingham et al., 1999,Science 284:1837-1841). Several studies have now demonstratedregeneration of hair cells in injured mice cochlea and improvement ofboth hearing and balance with virally mediated delivery of Math1 (Bakeret al., 2009, Adv. Otorhinolaryngol. 66:52-63; Husseman and Raphael,2009, Adv. Otorhinolaryngol. 66:37-51; Izumikawa et al., 2008, Hear.Res. 240:52-56; Kawamoto et al., 2003, J. Neurosci. 23:4395-4400;Praetorius et al., 2010, Acta Otolaryngol. 130:215-222; Staecker et al.,2007, Otol. Neurotol. 28:223-231).

Mutations in the WFS1 gene cause more than 90 percent of Wolframsyndrome type 1 cases; Wolfram syndrome is a condition that affects manyof the body's systems, most often characterized by high blood sugarlevels resulting from a shortage of the hormone insulin (diabetesmellitus) and progressive vision loss due to degeneration of the nervesthat carry information from the eyes to the brain (optic atrophy).However, people with Wolfram syndrome often also have pituitary glanddysfunction that results in the excretion of excessive amounts of urine(diabetes insipidus), hearing loss caused by changes in the inner ear(sensorineural deafness), urinary tract problems, reduced amounts of thesex hormone testosterone in males (hypogonadism), or neurological orpsychiatric disorders. About 65 percent of people with Wolfram syndromehave sensorineural deafness that can range in severity from deafnessbeginning at birth to mild hearing loss beginning in adolescence thatworsens over time. Furthermore, about 60 percent of people with Wolframsyndrome develop a neurological or psychiatric disorder, most commonlyproblems with balance and coordination (ataxia), typically beginning inearly adulthood.

The WFS1 gene encodes a protein called wolframin thought to regulate theamount of calcium in cells. When Wolfram syndrome is caused by mutationsin the WFS1 gene, it is inherited in an autosomal recessive pattern, andthe wolframin protein has reduced or absent function. As a result,calcium levels within cells are not regulated and the endoplasmicreticulum does not work correctly. When the endoplasmic reticulum doesnot have enough functional wolframin, the cell triggers its own celldeath (apoptosis). The death of cells in the pancreas, specificallycells that make insulin (beta cells), causes diabetes mellitus in peoplewith Wolfram syndrome. The gradual loss of cells along the optic nerveeventually leads to blindness in affected individuals. The death ofcells in other body systems likely causes the various signs and symptomsof Wolfram syndrome type 1.

Mutations in the mitochondrial genes MT-TS1 and MT-RNR1 have been foundto increase the risk of developing nonsyndromic deafness. Nonsyndromicmitochondrial hearing loss and deafness is characterized bymoderate-to-profound hearing loss. Pathogenic variants in MT-TS1 areusually associated with childhood onset of sensorineural hearing loss.Pathogenic variants in MT-RNR1 are associated with predisposition tohearing loss if they are exposed to certain antibiotic medicationscalled aminoglycosides (ototoxicity) and/or late-onset sensorineuralhearing loss; however, some people with a mutation in the MT-RNR1 genedevelop hearing loss even without exposure to these antibiotics. Hearingloss associated with aminoglycoside ototoxicity is bilateral and severeto profound, occurring within a few days to weeks after administrationof any amount (even a single dose) of an aminoglycoside antibiotic suchas gentamycin, tobramycin, amikacin, kanamycin, or streptomycin.

Treatments for hearing loss currently consist of hearing amplificationfor mild to severe losses and cochlear implantation for severe toprofound losses (Kral and O'Donoghue, 2010, N. Engl. J. Med.363:1438-1450). To date, a majority of the research in this arena hasfocused on cochlear hair cell regeneration, applicable to the mostcommon forms of hearing loss, including presbycusis, noise damage,infection, and ototoxicity.

In animal models for cochlear ischemia, ischemic damage may be preventedby compounds such as insulin-like growth factor (IGF-1), AM-111 (anapoptosis inhibitor), edarabone (a free radical scavenger), ginsenosideRB 1 (Kappo), glia-cell derived neurotrophic factor (GDNF), BDNF, CNTF,SOD1, SOD2, Necrostatin-1, DFNA5 and MSRB3. However, it appears that acombination of substances might be more effective than a single compound(e.g. complementary therapies to modulate oxidative stress, exotoxicity,blood flow, calcium and stimulation overload, apoptotic pathways,neurotrophic or hormonal control mechanisms).

Inhibition of JNK-1 induced apoptosis (mitochondria-induced) may beprevented by compounds such as dominant-negative JNK-1 and d-steroisomerJNK-1 (Mol. Pharmacol. 2007 March; 71(3):654-66; the contents of whichare herein incorporated by reference in its entirety).

A long-felt need remains for agents and methods for preventing orreversing deafness.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exemplary schematic representation of a genetic map of the5′ and 3′ vectors for dual-AAV transduction in inner hair cells (IHCs)using the trans-splicing approach. In the 5′ vector, a CMV enhancer(CMVe) and a human β-actin promoter (hbA) drive the transcription of anmRNA coding for eGFP and a P2A peptide, which is cleaved duringtranslation. The 5′ vector also contains cDNA encoding an N-terminalportion of otoferlin and a splice donor site (SD). The SD DNA sequencewas provided by Trapani et al. (2014) EMBO Mol Med 6 194-211. In the 3′vector, a splice acceptor site (SA) was subcloned after the firstinverted terminal repeat (ITR) and before the 3′ part of the codingsequence of otoferlin. This is followed by mRNA stabilizing sequences,woodchuck hepatitis virus posttranscriptional regulatory element (WPRE),and the poly-adenylation signal (pA).

FIG. 2 is an exemplary schematic representation of a genetic map of the5′ and 3′ vectors for dual-AAV transduction in IHCs, using the hybridapproach. In addition to the elements described in FIG. 1 , highlyrecombinogenic sequences from F1 phage (AK) sequences were included atthe 3‘ end of the’5 vector and at the 5′ end of the 3′ vector to forcecorrect vector assembly (Trapani et al. (2014) EMBO Mol Med 6 194-211).

FIG. 3 is a graph showing auditory brainstem responses (ABR) amplitudeover time in non-transduced Otof^(−/−) mice (n=25; thin black line) andOtof^(−/−) mice after dual-AAV mediated expression of otoferlin (n=14;grey lines represent individual animals, thick black line is the averageresponse across all animals).

FIG. 4 is a graph showing ABR amplitude over time in non-transducedOtof^(−/−) mice (n=25; long dashed line), Otof^(−/−) mice after dual-AAVmediated expression of otoferlin (n=14; short dashed line) and wild typeOtof^(wt/wt) mice after dual-AAV with enhanced green fluorescent protein(eGFP) transduction (n=5; thin black lines represent individual animals,thick black line is the average amplitude across all animals).

FIG. 5 is an exemplary graph showing ABR threshold (in dB SPL) overfrequency (in kHz) in response to pure tones or click stimuli. Theminimal sound pressure level (SPL) at which waves can be detected isdisplayed for individual Otof^(−/−) animals with dual-AAV mediatedexpression of otoferlin (white circles represent individual animals,circle with diagonal line fill is the average threshold across allanimals), for non-transduced Otof^(−/−) animals (black circles), and forOtof^(wt/wt) animals after dual-AAV with eGFP transduction (circle withcheckered fill), in response to pure tones or click stimuli.

FIG. 6 is a graph showing the transduction rates of IHCs along thecochlea (e.g., entire Corti, apical turn, and midbasal/basal turn),determined by immunohistochemistry using two different antibodies, onebinding the N-terminal otoferlin fragment, and the other binding to thevery C-terminal part of otoferlin.

FIG. 7 is a set of immunohistochemical images of one organ of Corti froman Otof^(−/−) mouse after dual-AAV mediated expression of otoferlin.Calbindin was used as marker for inner and outer hair cells. Cellsexpressing eGFP indicate virus transduction of at least the 5′ virus.Left and middle panels show anti-otoferlin antibody staining (Abcam) inthe N-terminal half of otoferlin. Right panel shows C-terminalanti-otoferlin antibody staining (Synaptic Systems). Together, all threepanels demonstrate that full-length otoferlin is expressed in IHCs.Importantly, despite the AAVs transduce several cell types in the organof Corti (indicated by eGFP fluorescence), otoferlin expression wasrestricted to inner hair cells. Scale bar: 100 μm.

FIG. 8 is a graph showing Ca²⁺ currents over the depolarization stimulusfor Otof^(−/−) IHCs (white; n=10), Otof^(−/−) IHC after dual-AAVmediated expression of otoferlin (diagonal line; n=8), and wild-typeIHCs from background matched control animals (black; n=6).

FIG. 9 is a graph showing plasma membrane capacitance (ΔC_(m)) over theduration of depolarization in dual-AAV transduced IHCs of Otof^(−/−),mice (black line, individual IHCs; diamond with diagonal line fill:mean±s.e.m; n=8), two individual dual-AAV-transduced IHCs from injectedOtof^(−/−) mice (dashed line), which displayed only minor amounts ofexocytosis, and background-matched wild-type controls (black diamonds;n=6). Wild-type data from Strenzke et al. (2016) EMBO J. 35 2519-2535 isshown with black circles for comparison.

FIG. 10 is a representative graph showing Qreal over the duration ofdepolarization (ms) in Otof^(−/−) IHCs (white diamond), dual-AAVtransduced IHCs of Otof^(−/−) mice (black circle), andbackground-matched wild-type controls (black diamond).

FIG. 11 is a representative plasmid map of pAAV-AK-SA-3′mOTOF-EWB.

FIG. 12 is a representative plasmid map of pAAV-SA-3′mOTOF-EWB.

FIG. 13 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5′mOTOF-SD.

FIG. 14 is a representative plasmid map ofpAAV-HBA-eGFP-P2A-5′mOTOF-SD-AK.

FIG. 15 is a graph showing plasma membrane capacitance (ΔCm) over theduration of depolarization in IHCs of Otof^(−/−) mice transduced withthe two vectors shown in FIG. 2 (medium shading; thick line), IHCs inwildtype mice (dark shading; medium thickness line), and IHCs inOtof^(−/−) mice.

FIG. 16 is a representative graph showing Qreal over the duration ofdepolarization (ms) in Otof^(−/−) IHCs (white diamond), IHCs ofOtof^(−/−) mice transduced with the two vectors shown in FIG. 2 (graydiamonds), and IHCs of background-matched wild-type controls (blackdiamond).

FIG. 17 is a representative plasmid map of pAAV-AK-SA-3′mOTOF-EWB.

FIG. 18 is a representative plasmid map of pAAV-HBA-eGFP-P2A-5′mOTOF-SD.

FIG. 19 is a representative plasmid map ofpAAV-HBA-eGFP-P2A-5′mOTOF-SD-AK.

FIG. 20 is a representative plasmid map of pAAV-SA-3′mOTOF-EWB.

FIG. 21 is a representative schematic of a portion of pAKOS102 (SEQ IDNO: 43).

FIG. 22 is a representative schematic of a portion of pAKOS103 (SEQ IDNO: 44).

FIG. 23 is a representative schematic of a portion of pAKOS103.

FIG. 24 is a representative plasmid map of pAKOS104 (SEQ ID NO: 45).

FIG. 25 is a representative schematic of a portion of pAKOS104-DHFR (SEQID NO: 46).

FIG. 26 is a representative schematic of a portion of pAKOS104-DHFR.

FIG. 27 is a representative schematic of a portion of pAKOS105 (SEQ IDNO: 47).

FIG. 28 is a representative schematic of a portion of pAKOS105.

FIG. 29 is a representative schematic of a portion of pAKOS105_GFP (SEQID NO: 48).

FIG. 30 is a representative schematic of a portion of pAKOS106 (SEQ IDNO: 49).

FIG. 31 is a representative schematic of a portion of pAKOS106.

FIG. 32 is a representative schematic of a portion of pAKOS107 (SEQ IDNO: 50).

FIG. 33 is a representative schematic of a portion of pAKOS107.

FIG. 34 is a representative schematic of a portion of pAKOS108 (SEQ IDNO: 51).

FIG. 35 is a representative schematic of a portion of pAKOS108.

FIG. 36 is a representative schematic of a portion of pAKOS109 (SEQ IDNO: 52).

FIG. 37 is a representative schematic a factor VIII stuffer (SEQ ID NOs.54-57).

FIG. 38 is a representative schematic of p109 (SEQ ID NO: 84).

FIG. 39 is a representative schematic of p105 (SEQ ID NO: 85).

FIG. 40 is a representative schematic of 105.WPRE.

FIG. 41 is a representative schematic of p108.

FIG. 42 is a representative schematic of 1OTOF18.CL1.

FIG. 43 is a representative schematic of 19OTOF48.

FIG. 44 is a representative schematic of 1OTOF20.CL1.

FIG. 45 is a representative schematic of 21OTOF48.WPRE.

FIG. 46 is a representative schematic of 1OTOF21.CL1.

FIG. 47 is a representative schematic of 22OTOF48.WPRE.

FIG. 48 is a representative schematic of 105.pA.NTF3.CMVd.

FIG. 49 is an immunoblot showing the expression of full-length humanotoferlin in HEK293FT cells transfected with the different pairs ofplasmids indicated.

FIG. 50 is a table showing the quantitation of expression of full-lengthhuman otoferlin from three replicates of the experiment described inFIG. 49 .

FIG. 51 is a graph of the click ABR threshold in wildtype not treatedwith a vector or Otof^(−/−) mice not treated with a vector or treatedwith DualAAV Anc80.hOtof vectors (p105 and p109 vectors). Hearing in theOtof^(−/−) mice administered the DualAAV Anc80.hOtof vectors (p105 and109 vectors) was measured at 26-28 days and 91 days after treatment.

FIG. 52 is a graph of the tone burst ABR threshold in wildtype nottreated with a vector or Otof^(−/−) mice not treated with a vector ortreated with DualAAV Anc80.hOtof vectors (p105 and p109 vectors).Hearing in the Otof^(−/−) mice administered the DualAAV Anc80.hOtofvectors (p105 and 109 vectors) was measured at 26-28 days and 91 daysafter treatment.

FIG. 53 is a representative schematic of a portion ofpAAV-HBA-eGFP-P2A-5′mOTOF.SD (SEQ ID NO: 87).

FIG. 54 is a representative schematic of a portion ofpAAV-SA-3′mOTOF.WPRE (SEQ ID NO: 88).

FIG. 55 is a representative schematic of a portion ofpAAV-HBA-eGFP-P2A-5′mOTOF.SD-AK.

FIG. 56 is a representative schematic of a portion ofpAAV-AK-SA-3′mOTOF.WPRE.

FIG. 57 is a representative schematic of a portion ofpAAV-CMV-5′hOTOF-SD-AK.

FIG. 58 is a representative schematic of a portion ofpAAV-HBA-5′hOTOF-SD-AP.

FIG. 59 is a representative schematic of a portion ofpAAV-HBA-5′hOTOF-SD-AK.

FIG. 60 is a representative schematic of a portion ofpAAV-HBA-5′hOTOFcodop-SD-AK.

FIG. 61 is a representative schematic of a portion ofpAAV-HBA-5′hOTOFcodop-SD.

FIG. 62 is a representative schematic of a portion ofpAAV-CMV-5′hOTOFcodop-SD.

FIG. 63 is a representative schematic of a portion ofpAAV-CMV-5′hOTOFcodop-SD-AK.

FIG. 64 is a representative schematic of a portion ofpAAV-CBA-5′hOTOFcodop-SD-AK.

FIG. 65 is a representative schematic of a portion ofpAAV-CBA-5′hOTOF-SD.

FIG. 66 is a representative schematic of a portion of pAAV-SA-3′OTOF.

FIG. 67 is a representative schematic of a portion of pAAV-AP-SA-3′OTOF.

FIG. 68 is a representative schematic of a portion ofpAAV-AK-SA-3′OTOFcodop.

FIG. 69 is a representative immunoblot showing the expression offull-length human otoferlin in HEK293FT cells transfected using DNAtransfection reagent jetPRIME® (polyplus) with 600 ng of the differentpairs of plasmids indicated. Lane 1 contained a prestained proteinladder. Lane 2 contained a protein sample of HEK293FT cells that weretransfected with vector pAKOS104 (as shown in FIGS. 24 and 59 ) andvector pAKOS105 (as shown in FIGS. 27, 28 and 39 ). Lane 3 contained aprotein sample of HEK293FT cells that were transfected with vectorpAKOS108 (as shown in FIGS. 34, 35, 41 and 57 ) and vector pAKOS105 (asshown in FIGS. 27, 28 and 39 ). Lane 4 contained a protein sample ofHEK293FT cells that were transfected with vector pAKOS109 (as shown inFIGS. 36 and 38 ) and vector pAKOS105 (as shown in FIGS. 27, 28 and 39). Lane 5 contained a protein sample of HEK293FT cells that weretransfected with vector pAAV-HBA-5′hOTOFcodop-SD-AK (as shown in FIG. 60) and vector pAAV-AK-SA-3′OTOFcodop (as shown in FIG. 68 ). Lane 6contained a protein sample of HEK293FT cells that were transfected withvector pAAV-CMV-5′hOTOFcodop-SD-AK (as shown in FIG. 63 ) and vectorpAAV-AK-SA-3′OTOFcodop (as shown in FIG. 68 ). Lane 7 contained aprotein sample of HEK293FT cells that were transfected with vectorpAAV_CBA-5′hOTOFcodop-SD-AK (as shown in FIG. 64 ) and vectorpAAV-AK-SA-3′OTOFcodop (as shown in FIG. 68 ). Lane 8 contained aprotein sample of HEK293FT cells that were transfected with vectorpAKOS102 (as shown in FIG. 21 ) and vector pAKOS103 (as shown in FIGS.22, 23 and 66 ). Lane 9 contained a protein sample of HEK293FT cellsthat were transfected with a CBA.TS vector and vector pAKOS103 (as shownin FIGS. 22, 23 and 66 ). Lane 10 contained a protein sample of HEK293FTcells that were transfected with vector pAKOS106 (as shown in FIGS. 30,31 and 58 ) and pAKOS107 (as shown in FIGS. 32, 33 and 67 ). Ninety-sixhours post-transfection, cells were harvested and lysed using RIPAbuffer and analyzed in 4-12% Bolt protein gel, which was thentransferred onto a nitrocellulose membrane. Human otoferlin was detectedusing an anti-OTOF polyclonal antibody (Thermo PA5-52935). Humanbeta-actin was used as the primary antibody for internal loading controlbetween lanes. The experiment was repeated in triplicate. Relativequantitative measurements for each experiment are provided under theimmunoblot, along with the average measurement and standard deviation(STDEV).

FIG. 70 is an immunoblot showing the expression of full-length humanotoferlin in HEK293FT cells transfected with the different pairs ofplasmids indicated at different multiplicity of infections (MOI).HEK293FT cells were seeded overnight at 4×10⁴ cells/well on a 96-wellplate. Six hours post-seeding, the dual vectors were added to each well.Ninety-six hours post-transfection, cells were harvested and lysed usingRIPA buffer and analyzed in 4-12% Bolt protein gel, which was thentransferred onto a nitrocellulose membrane. Human otoferlin was detectedusing an anti-OTOF polyclonal antibody (Thermo PA5-52935). Humanbeta-actin was used as the primary antibody for internal loading controlbetween lanes. Lane 1: CBA.OTOF(AK) with MOI 503,000; lane 2:CBA.OTOF(AK) with MOI 1,510,000; lane 3: CBA.OTOF(AK) with MOI 100,000;lane 4: CBA.OTOF(AK) with MOI 303,000; lane 5: CMV.OTOF(AK) with MOI638,000; lane 6: CMV.OTOF(AK) with MOI 1,910,000; lane 7: CMV.OTOF(AK)with MOI 127,000; lane 8: CMV.OTOF(AK) with MOI 382,000; lane 9:negative control.

FIG. 71 a set of immunohistochemical images of one organ of Corti froman Otof−/− mouse age P17 after unilateral intracochlear administrationof dual-AAV vectors expressing CBA.hOTOF(AK) (p105 and 109 vectors).Ipsilateral cochlea was dissected and analyzed for protein expressingusing immunohistochemistry at three different frequency regions(base-apex).

FIG. 72 is a graph showing the percentage of N- and C-terminal otoferlinlabeled inner hair cells (IHC) in dual-AAV-TS (n=10 mice) anddual-AAV-Hyb (n=9 mice) injected CD1B6F1-Otof^(−/−) mice (aged P18-P30).Individual animals are depicted with open symbols. Data are displayed asmean±standard error of mean (s.e.m.), ns P>0.05; *P≤0.05; **P≤0.01;***P≤0.001, Wilcoxon matched-pair signed rank test, and unpaired t-testwith Welch's correction.

FIG. 73 is a graph showing the average N-terminal and C-terminalotoferlin immunofluorescence levels in dual-AAV-transduced Otof^(−/−)and wild-type inner hair cells (IHC) from mice (aged P23-30). Otoferlinlevels were normalized to immunofluorescence levels in non-transduced B6wild-type IHCs for each antibody separately. The number of quantifiedIHCs is indicated inside the bars. Data are displayed as mean±standarderror of mean (s.e.m.), ns P>0.05; *P≤0.05; **P≤0.01; ***P≤0.001,Kruskal-Wallis test followed by Dunn's multiple comparison test.

FIG. 74 is a graph showing synaptic ribbon numbers quantified from innerhair cells (IHCs) in apical cochlear turns of wild-type (B6: n=48 IHCs,CD1B6F1: n=108 IHCs), transduced Otof^(−/−) (dualAAV-TS: n=59 IHCs,dualAAV-Hyb: n=37 IHCs), and non-transduced Otof^(−/−) IHCs frominjected (−AAV-injected ear, n=65 IHCs) and contralateral non-injected(−AAV non-injected ear, n=46 IHCs) ears (from mice aged P25-P29.Individual animals are depicted with open symbols. Data are displayed asmean±standard error of mean (s.e.m.), ns P>0.05, **P≤0.001(Kruskal-Wallis test followed by Dunn's multiple comparison test).

FIG. 75 is a graph showing synapse numbers quantified from inner haircells (IHCs) in apical turns (c) of B6 wild-type (P6: n=53 IHCs; P14:n=73 IHCs) and B6 Otof^(−/−) (P6: n=62 IHCs; P14: n=65 IHCs) mice at twodifferent developmental stages (P6 and P14). Individual animals aredepicted with open symbols. Data are displayed as mean±standard error ofmean (s.e.m.), ns P>0.05, **P≤0.001 (Kruskal-Wallis test followed byDunn's multiple comparison test).

FIG. 76 is a graph showing summed auditory brainstem response (ABR) waveI-V amplitudes at different click sound intensities in otoferlindual-AAV-injected, non-injected Otof^(−/−), and wild-type control miceaged P23-30. Number of analyzed mice: CD1B6F1 wild-type animals(+AAV.eGFP: n=12 mice, dualAAV-TS: n=6 mice) and CD1B6F1 Otof^(−/−)animals (dualAAV-TS: n=16 mice, dualAAV-Hyb: n=8 mice, =AAV: n=38 mice).Data are represented as mean±standard error of mean (s.e.m.) Individualanimals are depicted with open symbols.

FIG. 77 is a graph showing summer auditory brainstem response (ABR) waveI-V amplitudes of individual dual-AAV-TS treated CD1B6F1 Otof^(−/−),animals (n=8 animals; from FIG. 76 ) plotted against their full-lengthotoferlin inner hair cell (IHC) transduction rate (from FIG. 5 ,C-terminal otoferlin). r: correlation coefficient. =8 mice, =AAV: n=38mice). Individual animals are depicted with open symbols. r≥0.5 positivecorrelation (70 decibel of sound pressure level (dB SPL) and 90 dB SPL:Pearson correlation test; 50 dB SPL: Spearman correlation test).

FIG. 78 is a schematic of an exemplary dual AAV vector system of thepresent disclosure which includes “upstream” and “downstream” vectors(AKhOTOF5 and AKhOTOF3, respectively).

FIG. 79 is a schematic of the “upstream” vector AKhOTOF5.

FIG. 80 is a schematic of the “downstream” vector AKhOTOF3.

FIG. 81 illustrates a perspective of a device for delivering fluid to aninner ear, according to aspects of the present disclosure.

FIG. 82 illustrates a sideview of a bent needle sub-assembly, accordingto aspects of the present disclosure.

FIG. 83 illustrates a perspective view of a device for delivering fluidto an inner ear, according to aspects of the present disclosure.

FIG. 84 illustrates a perspective view of a bent needle sub-assemblycoupled to the distal end of a device, according to aspects of thepresent disclosure.

SUMMARY

The present disclosure is based on the discovery that a compositionincluding at least two different nucleic acid vectors, where each of theat least two different vectors includes a coding sequence that encodes adifferent portion of an otoferlin protein, can be used to generate asequence encoding an active otoferlin protein (e.g., a full-lengthotoferlin protein) in a mammalian cell, and thereby treat non-syndromicsensorineural hearing loss in a subject in need thereof.

Provided herein are compositions that include at least two differentnucleic acid vectors, wherein: each of the at least two differentvectors includes a coding sequence that encodes a different portion ofan otoferlin protein, each of the encoded portions being at least 30amino acid residues in length, wherein the amino acid sequence of eachof the encoded portions may optionally partially overlap with the aminoacid sequence of a different one of the encoded portions; no singlevector of the at least two different vectors encodes a full-lengthotoferlin protein; at least one of the coding sequences includes anucleotide sequence spanning two neighboring exons of otoferlin genomicDNA, and lacks an intronic sequence between the two neighboring exons;and when introduced into a mammalian cell the at least two differentvectors undergo concatemerization or homologous recombination with eachother, thereby forming a recombined nucleic acid that encodes afull-length otoferlin protein. In some embodiments of any of thecompositions described herein, each of the at least two differentvectors is a plasmid, a transposon, a cosmid, an artificial chromosome,or a viral vector. In some embodiments of any of the compositionsdescribed herein, each of the at least two different vectors is a humanartificial chromosome (HAC), yeast artificial chromosome (YAC),bacterial artificial chromosome (BAC), or a P1-derived artificialchromosome (PAC). In some embodiments of any of the compositionsdescribed herein, each of the at least two different vectors is a viralvector selected from an adeno-associated virus (AAV) vector, anadenovirus vector, a lentivirus vector, or a retrovirus vector. In someembodiments of any of the compositions described herein, each of the atleast two different vectors is an AAV vector.

In some embodiments of any of the compositions described herein, theamino acid sequence of one of the encoded portions overlaps with theamino acid sequence of a different one of the encoded portions. In someembodiments of any of the compositions described herein, the amino acidsequence of each of the encoded portions partially overlaps with theamino acid sequence of a different encoded portion. In some embodimentsof any of the compositions described herein, the overlapping amino acidsequence is between about 30 amino acid residues to about 1000 aminoacid residues in length.

In some embodiments of any of the compositions described herein, thevectors include two different vectors, each of which includes adifferent segment of an intron, wherein the intron includes thenucleotide sequence of an intron that is present in otoferlin genomicDNA, and wherein the two different segments overlap in sequence by atleast 100 nucleotides. In some embodiments of any of the compositionsdescribed herein, the two different segments overlap in sequence byabout 100 nucleotides to about 800 nucleotides. In some embodiments ofany of the compositions described herein, the nucleotide sequence ofeach of the at least two different vectors is between about 500nucleotides to about 10,000 nucleotides in length. In some embodimentsof any of the compositions described herein, the nucleotide sequence ofeach of the at least two different vectors is between 500 nucleotides to5,000 nucleotides in length.

In some embodiments of any of the compositions described herein, thenumber of different vectors in the composition is two. In someembodiments of any of the compositions described herein, a first of thetwo different vectors includes a coding sequence that encodes anN-terminal portion of the otoferlin protein. In some embodiments of anyof the compositions described herein, the N-terminal portion of theotoferlin protein is between 30 amino acids to 1600 amino acids inlength. In some embodiments of any of the compositions described herein,the N-terminal portion of the otoferlin protein is between 200 aminoacids to 1500 amino acids in length. In some embodiments of any of thecompositions described herein, the first vector further includes one orboth of a promoter and a Kozak sequence. In some embodiments of any ofthe compositions described herein, the first vector includes a promoterthat is an inducible promoter, a constitutive promoter, or atissue-specific promoter.

In some embodiments of any of the compositions described herein, one ofthe two vectors comprises SEQ ID NO: 39 (or comprises a sequence that isat least 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 98%, or atleast 99% identical to SEQ ID NO: 39) and the second of the two vectorscomprises SEQ ID NO: 40 (or comprises a sequence that is at least 80%,at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 98%, or at least 99%identical to SEQ ID NO: 40). In some embodiments of any of thecompositions described herein, one of the two vectors comprises SEQ IDNO: 41 (or comprises a sequence that is at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, or at least 99% identical to SEQID NO: 41) and the second of the two vectors comprises SEQ ID NO: 42 (orcomprises a sequence that is at least 80%, at least 82%, at least 84%,at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, or at least 99% identical to SEQ ID NO: 42). Insome embodiments of any of the compositions described herein, one of thetwo vectors comprises SEQ ID NO:84 (or comprises a sequence that is atleast 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 98%, or atleast 99% identical to SEQ ID NO: 84) and the second of the two vectorscomprises SEQ ID NO: 85 (or comprises a sequence that is at least 80%,at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 98%, or at least 99%identical to SEQ ID NO: 85).

In some embodiments of any of the compositions described herein, whereinone of the at least two different vectors comprises a sequence encodinga NTF3 protein.

In some embodiments of any of the compositions described herein, whereinthe sequence encoding a NTF3 protein is at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, or at least 99% identical to SEQID NO: 78.

In some embodiments of any of the compositions described herein, thefirst vector further includes a coding sequence encoding adestabilization domain, wherein the destabilization domain is 3′ to thecoding sequence that encodes the N-terminal portion of the otoferlinprotein.

In some embodiments of any of the compositions described herein, thecoding sequence that encodes the N-terminal portion of the otoferlinprotein comprises exons 1-21 of isoform 5 of the human otoferlin gene.

In some embodiments of any of the compositions described herein, thesecond of the two different vectors includes a coding sequence thatencodes a C-terminal portion of the otoferlin protein. In someembodiments of any of the compositions described herein, the C-terminalportion of the otoferlin protein is between 30 amino acids to 1600 aminoacids in length. In some embodiments of any of the compositionsdescribed herein, the C-terminal portion of the otoferlin protein isbetween 200 amino acids to 1500 amino acids in length. In someembodiments of any of the compositions described herein, the secondvector further includes a poly(dA) signal sequence. In some embodimentsof any of the compositions described herein, the coding sequence thatencodes the C-terminal portion of the otoferlin protein comprises exons22-48 of isoform 5 of the human otoferlin gene. In some embodiments ofany of the compositions described herein, the second vector furtherincludes sequences for mRNA stabilization. Some embodiments of any ofthe compositions described herein further include a pharmaceuticallyacceptable excipient.

Also provided herein are kits that include any of the compositionsdescribed herein. Some embodiments of any of the kits described hereinfurther include a pre-loaded syringe containing the composition.

Also provided herein are methods that include introducing into a cochleaof a mammal a therapeutically effective amount of any of thecompositions described herein. In some embodiments of any of thecompositions described herein, the mammal is a human. In someembodiments of any of the compositions described herein, the mammal hasbeen previously identified as having a defective otoferlin gene.

Also provided herein are methods of increasing expression of an activeotoferlin protein, e.g., a full-length otoferlin protein, in a mammaliancell that include introducing any of the compositions described hereininto the mammalian cell. In some embodiments of any of the methodsdescribed herein, the mammalian cell is a cochlear inner hair cell. Insome embodiments of any of the methods described herein, the mammaliancell is a human cell. In some embodiments of any of the methodsdescribed herein, the mammalian cell has previously been determined tohave a defective otoferlin gene.

Also provided herein are methods of increasing expression of an activeotoferlin protein, e.g., a full-length otoferlin protein in an innerhair cell in a cochlea of a mammal that include: introducing into thecochlea of the mammal a therapeutically effective amount of any of thecompositions described herein. In some embodiments of any of the methodsdescribed herein, the mammal has been previously identified as having adefective otoferlin gene. In some embodiments of any of the methodsdescribed herein, the mammal is a human.

Also provided herein are methods of treating non-syndromic sensorineuralhearing loss in a subject identified as having a defective otoferlingene that include administering a therapeutically effective amount of acomposition of any one of the compositions described herein into thecochlea of the subject. In some embodiments of any of the methodsdescribed herein, the subject is a human. Some embodiments of any of themethods described herein further include, prior to the administeringstep, determining that the subject has a defective otoferlin gene.

Also provided herein are compositions that include two different nucleicacid vectors, wherein: a first nucleic acid vector of the two differentnucleic acid vectors includes a promoter, a first coding sequence thatencodes an N-terminal portion of an otoferlin protein positioned 3′ ofthe promoter, and a splicing donor signal sequence positioned at the 3′end of the first coding sequence; and a second nucleic acid vector ofthe two different nucleic acid vectors includes a splicing acceptorsignal sequence, a second coding sequence that encodes a C-terminalportion of an otoferlin protein positioned at the 3′ end of the splicingacceptor signal sequence, and a polyadenylation sequence at the 3′ endof the second coding sequence; wherein each of the encoded portions isat least 30 amino acid residues in length, wherein the amino acidsequences of the encoded portions do not overlap, wherein no singlevector of the two different vectors encodes a full-length otoferlinprotein, and, when the coding sequences are transcribed in a mammaliancell, to produce RNA transcripts, splicing occurs between the splicingdonor signal sequence on one transcript and the splicing acceptor signalsequence on the other transcript, thereby forming a recombined RNAmolecule that encodes a full-length otoferlin protein. In someembodiments of any of the compositions described herein, the codingsequence of at least one of the vectors includes a nucleotide sequencespanning two neighboring exons of otoferlin genomic DNA, and lacks anintronic sequence between the two neighboring exons.

Also provided herein are compositions that include: a first nucleic acidvector including a promoter, a first coding sequence that encodes anN-terminal portion of an otoferlin protein positioned 3′ of thepromoter, a splicing donor signal sequence positioned at the 3′ end ofthe first coding sequence, and a first detectable marker gene positioned3′ of the splicing donor signal sequence; and a second nucleic acidvector, different from the first nucleic acid vector, including a seconddetectable marker gene, a splicing acceptor signal sequence positioned3′ of the second detectable marker gene, a second coding sequence thatencodes a C-terminal portion of an otoferlin protein positioned at the3′ end of the splicing acceptor signal sequence, and a polyadenylationsequence positioned at the 3′ end of the second coding sequence; whereineach of the encoded portions is at least 30 amino acid residues inlength, wherein the respective amino acid sequences of the encodedportions do not overlap with each other, wherein no single vector of thetwo different vectors encodes a full-length otoferlin protein, and, whenthe coding sequences are transcribed in a mammalian cell to produce RNAtranscripts, splicing occurs between the splicing donor signal on onetranscript and the splicing acceptor signal on the other transcript,thereby forming a recombined RNA molecule that encodes a full-lengthotoferlin protein. In some embodiments of any of the compositionsdescribed herein, the coding sequence of at least one of the vectorsincludes a nucleotide sequence spanning two neighboring exons ofotoferlin genomic DNA, and lacks an intronic sequence between the twoneighboring exons. In some embodiments of any of the compositionsdescribed herein, the first or second detectable marker gene encodesalkaline phosphatase. In some embodiments of any of the compositionsdescribed herein, the first and second detectable marker genes are thesame.

Also provided herein are compositions that include a first nucleic acidvector including a promoter, a first coding sequence that encodes anN-terminal portion of an otoferlin protein positioned 3′ to thepromoter, a splicing donor signal sequence positioned at the 3′ end ofthe first coding sequence, and a highly recombinogenic sequence (e.g., aF1 phage recombinogenic region, e.g., SEQ ID NO: 66) positioned 3′ tothe splicing donor signal sequence; and a second nucleic acid vector,different from the first nucleic acid vector, including a second highlyrecombinogenic sequence (e.g., a F1 phage recombinogenic region, e.g.,SEQ ID NO: 67, or an alkaline phosphatase recombinogenic region, e.g.,SEQ ID NO: 89), a splicing acceptor signal sequence positioned 3′ of thesecond highly recombinogenic sequence (e.g., a F1 phage recombinogenicregion or an alkaline phosphatase recombinogenic region), a secondcoding sequence that encodes a C-terminal portion of an otoferlinprotein positioned at the 3′ end of the splicing acceptor signalsequence, and a polyadenylation sequence positioned at the 3′ end of thesecond coding sequence; wherein each of the encoded portions is at least30 amino acid residues in length, wherein the respective amino acidsequences of the encoded portions do not overlap with each other,wherein no single vector of the two different vectors encodes afull-length otoferlin protein, and, when the coding sequences aretranscribed in a mammalian cell to produce RNA transcripts, splicingoccurs between the splicing donor signal one transcript and the splicingacceptor signal on the other transcript, thereby forming a recombinedRNA molecule that encodes a full-length otoferlin protein.

In some embodiments of any of the compositions described herein, thecoding sequence of at least one of the vectors includes a nucleotidesequence spanning two neighboring exons of otoferlin genomic DNA, andlacks an intronic sequence between the two neighboring exons.

Also provided herein are kits that include any of the compositionsdescribed herein. Some embodiments of any of the kits described hereinfurther include a pre-loaded syringe containing the composition.

Also provided herein are methods that include introducing into a cochleaof a mammal a therapeutically effective amount of any of thecompositions described herein. In some embodiments of any of the methodsdescribed herein, the mammal is a human. In some embodiments of any ofthe methods described herein, the mammal has been previously identifiedas having a defective otoferlin gene.

Also provided herein are methods of increasing expression of afull-length otoferlin protein in a mammalian cell that includeintroducing any of the compositions described herein into the mammaliancell. In some embodiments of any of the methods described herein, themammalian cell is a cochlear inner hair cell. In some embodiments of anyof the methods described herein, the mammalian cell is a human cell. Insome embodiments of any of the methods described herein, the mammaliancell has previously been determined to have a defective otoferlin gene.

Also provided herein are methods of increasing expression of afull-length otoferlin protein in an inner hair cell in a cochlea of amammal that include introducing into the cochlea a therapeuticallyeffective amount of any of the compositions described herein. In someembodiments of any of the methods described herein, the mammal has beenpreviously identified as having a defective otoferlin gene. In someembodiments of any of the methods described herein, the mammal is ahuman.

Also provided herein are methods of treating non-symptomaticsensorineural hearing loss in a subject identified as having a defectiveotoferlin gene that include administering a therapeutically effectiveamount of any of the compositions described herein into a cochlea of thesubject. In some embodiments of any of the methods described herein, thesubject is a human. Some embodiments of any of the methods describedherein further include, prior to the administering step, determiningthat the subject has a defective otoferlin gene.

Provided herein are therapeutic compositions including a plurality ofadeno-associated viral (AAV) vectors, wherein the plurality of AAVvectors are capable of constituting an auditory polypeptide messengerRNA in a target cell of a human subject to whom the therapeuticcomposition is administered.

In some embodiments of any of the therapeutic compositions describedherein, the plurality of AAV vectors are capable of constituting afull-length auditory polypeptide messenger RNA in a target cell of ahuman subject to whom the therapeutic composition is administered.

Some embodiments of any of the therapeutic composition described hereinfurther include a first AAV vector and a second AAV vector, wherein thefirst and second AAV vectors independently contain packaging capacity ofless than about 6 kb.

In some embodiments of any of the therapeutic compositions describedherein, the auditory polypeptide messenger RNA encodes an auditorypolypeptide selected from the group of otoferlin and an ortholog orhomolog thereof.

Some embodiments of any of the therapeutic compositions describedherein, can further include a nucleic acid (e.g., a vector) including anucleic acid sequence encoding an auditory polypeptide messenger RNAencodes an auditory polypeptide selected from the group consisting ofCa_(v)1.3, a scaffold protein selected from bassoon, piccolo, ribeye,and harmonin, Vglut3, synaptotagmin, a vesicle tethering/dockingprotein, a vesicle priming protein, a vesicle fusion proteins, GluA2/3,and GluA4.

In some embodiments of any of the therapeutic compositions describedherein, the first AAV vector further includes at least one promotersequence selected from a CBA, a CMV, or a CB7 promoter.

In some embodiments of any of the therapeutic compositions describedherein, the first AAV vector further includes at least one promotersequence selected from Cochlea-specific promoters.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated for intra-cochlearadministration. In some embodiments of any of the therapeuticcompositions described herein, the therapeutic composition is formulatedto include a lipid nanoparticle. In some embodiments of any of thetherapeutic compositions described herein, the therapeutic compositionis formulated to include a polymeric nanoparticle. In some embodimentsof any of the therapeutic compositions described herein, the therapeuticcomposition is formulated to include a mini-circle DNA. In someembodiments of any of the therapeutic compositions described herein, thetherapeutic composition is formulated to include a CELiD DNA. In someembodiments of any of the compositions described herein, the therapeuticcomposition is formulated to include a synthetic perilymph solution. Insome embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a syntheticperilymph solution including 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mMCaCl₂); 1-10 mM glucose; and 2-50 mM HEPES; and having a pH of betweenabout 6 and about 9.

Also provided herein are therapeutic compositions that include anauditory polypeptide messenger RNA.

Also provided herein are therapeutic compositions that include one or aplurality of adenoviral (AV) vectors, where the one or the plurality ofAV vectors are capable of constituting an auditory polypeptide messengerRNA in a target cell of a human subject to whom the therapeuticcomposition is administered.

In some embodiments of any of the therapeutic compositions describedherein, the one or the plurality of AV vectors are capable ofconstituting a full-length auditory polypeptide messenger RNA in atarget cell of a human subject to whom the therapeutic composition isadministered.

Also provided herein are therapeutic compositions including one or aplurality of lentiviral vectors, where the one or the plurality oflentiviral vectors are capable of constituting an auditory polypeptidemessenger RNA in a target cell of a human subject to whom thetherapeutic composition is administered.

In some embodiments of any of the therapeutic compositions describedherein, the one or the plurality of lentiviral vectors are capable ofconstituting an active, e.g., full-length auditory polypeptide messengerRNA in a target cell of a human subject to whom the therapeuticcomposition is administered.

Also provided herein are surgical methods that include the steps of: i)introducing into a cochlea of a human subject a first incision at afirst incision point; ii) administering intra-cochlearly an effectivedose of a therapeutic composition (e.g., any of the therapeuticcompositions described herein).

In some embodiments of any of the methods described herein, thetherapeutic composition is administered to the subject at the firstincision point. In some embodiments of any of the methods describedherein, the therapeutic composition is administered to the subject intoor through the first incision.

In some embodiments of any of the methods described herein, thetherapeutic composition is administered to the subject into or throughthe cochlea oval window membrane. In some embodiments of any of themethods described herein, the therapeutic composition is administered tothe subject into or through the cochlea round window membrane.

In some embodiments of any of the methods described herein, thetherapeutic composition is administered using a medical device capableof creating a plurality of incisions in the round window membrane.

In some embodiments of any of the methods described herein, the medicaldevice includes a plurality of micro-needles. In some embodiments of anyof the methods described herein, the medical device includes a pluralityof micro-needles including a generally circular first aspect, where eachmicro-needle has a diameter of at least about 10 microns.

In some embodiments of any of the methods described herein, the medicaldevice includes a base and/or a reservoir capable of holding thetherapeutic composition. In some embodiments of any of the methodsdescribed herein, the medical device includes a plurality of hollowmicro-needles individually including a lumen capable of transferring thetherapeutic composition.

In some embodiments of any of the methods described herein, the medicaldevice includes a means for generating at least a partial vacuum.

Also provided herein are therapeutic delivery systems, that include i) amedical device capable of creating a plurality of incisions in a roundwindow membrane of an inner ear of a human subject in need thereof, andii) an effective dose of a therapeutic composition including a pluralityof adeno-associated viral (AAV) vectors, wherein the plurality of AAVvectors are capable of constituting an active, e.g., full-length,auditory polypeptide messenger RNA in a target cell of the inner ear.

Also provided herein are means for performing a surgical method thatincludes the steps of: i) administering intra-cochlearly to a humansubject in need thereof an effective dose of the therapeutic composition(e.g., any of the therapeutic composition described herein), where thetherapeutic composition is capable of being administered by using amedical device including a) means for creating a plurality of incisionsin the round window membrane and b) the effective dose of thetherapeutic composition.

In some embodiments of any of the means for performing a surgical methoddescribed herein, the medical device includes a plurality ofmicro-needles.

Also provided herein are therapeutic compositions that include a singleadeno-associated viral (AAV) vector, where the AAV vector is capable ofconstituting an auditory polypeptide messenger RNA in a target cell of ahuman subject to whom the therapeutic composition is administered.

In some embodiments of any of the therapeutic compositions describedherein, the single AAV vector is capable of constituting a full-lengthauditory polypeptide messenger RNA in a target cell of a human subjectto whom the therapeutic composition is administered. In some embodimentsof any of the methods described herein, a single vector (e.g., any ofthe vectors described herein) that includes a sequence encoding anactive otoferlin protein (e.g., any of the full-length or truncatedactive otoferlin proteins described herein) can be administered to thesubject.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition further includes a second vectorother than an AAV vector, where the single AAV vector and the secondvector independently contain packaging capacity of less than about 6 kb.

In some embodiments of any of the therapeutic compositions describedherein, the auditory polypeptide messenger RNA encodes an auditorypolypeptide selected from the group of otoferlin and an ortholog orhomolog thereof.

In some embodiments of any of the therapeutic compositions describedherein, the auditory polypeptide messenger RNA encodes an auditorypolypeptide selected from the group of otoferlin and truncation mutantthereof.

In some embodiments of any of the therapeutic compositions describedherein, the otoferlin truncation mutant includes at least a single C2domain of the following:

a.a. Designation (from Pangrsic et al. 2012 C2 Domain Trends Neurosci35(11):671-680 C2A  1-121 C2B 256-378 C2C 419-542 C2D  962-1095 C2E1494-1622 C2F 1734-1895

In some embodiments of any of the therapeutic compositions describedherein, the otoferlin truncation mutant does not include an endogenousotoferlin polypeptide C-terminal region.

In some embodiments of any of the therapeutic compositions describedherein, the auditory polypeptide messenger RNA encodes an auditorypolypeptide selected from the group consisting of Ca_(v)1.3, a scaffoldprotein selected from bassoon, piccolo, ribeye, and harmonin, Vglut3,synaptotagmin, a vesicle tethering/docking protein, a vesicle primingprotein, a vesicle fusion proteins, GluA2/3, and GluA4.

In some embodiments of any of the therapeutic compositions describedherein, the single AAV vector further includes at least one promotersequence selected from a CBA, a CMV, or a CB7 promoter.

In some embodiments of any of the therapeutic compositions describedherein, the single AAV vector further includes at least one promotersequence selected from a Cochlea-specific promoters.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated for intra-cochlearadministration.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a lipidnanoparticle.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a polymericnanoparticle.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include amini-circle DNA.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a CELiDDNA.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a syntheticperilymph solution.

In some embodiments of any of the therapeutic compositions describedherein, the therapeutic composition is formulated to include a syntheticperilymph solution including 20-200 mM NaCl; 1-5 mM KCl; 0.1-10 mMCaCl₂); 1-10 mM glucose; 2-50 mM HEPES; having a pH of between about 6and about 9.

Also provided herein are therapeutic compositions that include anauditory polypeptide messenger RNA encoding an otoferlin truncationmutant.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

The terms “a” and “an” refer to one or to more than one (i.e., at leastone) of the grammatical object of the article. By way of example, “anelement” encompasses one element and more than one element.

The term “about” when referring to a measurable value such as an amount,a temporal duration, and the like, is meant to encompass variations ofup to ±20%. These variations can be, for example, up to ±10%, or in someinstances ±5%, or in some instances ±1%, or in some instances ±0.1% fromthe specified value, as such variations are appropriate to perform thedisclosed methods.

The term “signaling domain” refers to the functional portion of aprotein which acts by transmitting information within the cell toregulate cellular activity via defined signaling pathways by generatingsecond messengers or functioning as effectors by responding to suchmessengers. Otoferlin is believed to be a calcium sensor required forexocytosis in inner hair cells, as well as neurotransmitter release inimmature outer hair cells. Five of the six C2 domains of the otoferlinprotein (C2B-F) bind calcium ions with moderate and low affinityconstants in solution (Kd=25-95 μM and 400-700 μM, respectively)(Padmanarayana et al. 2014 Biochem 53:5023-5033). In the presence ofphosphatidylserine (PS), calcium concentrations of 10 μM result insignificant C2-liposome interaction for the C2C-C2E domains ofotoferlin. Thus, otoferlin possesses domains that appear to operateusing an “electrostatic switch” mechanism, as well as domains that bindregardless of calcium. PI(4,5)P2, a major signaling molecule at thepresynapse, has been shown to interact with the C2C and C2F domains ofotoferlin in a calcium-independent fashion (Padmanarayana et al. 2014Biochem 53:5023-5033).

The term “antibody,” as used herein, refers to a protein, or polypeptidesequence derived from an immunoglobulin molecule, which specificallybinds with an antigen. Antibodies can be polyclonal or monoclonal,multiple or single chain, or intact immunoglobulins, and may be derivedfrom natural sources or from recombinant sources. Antibodies can betetramers of immunoglobulin molecules. The term “antibody fragment”refers to at least one portion of an intact antibody, or recombinantvariants thereof, and refers to the antigen binding domain, e.g., anantigenic determining variable region of an intact antibody, that issufficient to confer recognition and specific binding of the antibodyfragment to a target, such as an antigen. Examples of antibody fragmentsinclude, but are not limited to, Fab, Fab′, F(ab′)₂, and Fv fragments,scFv antibody fragments, linear antibodies, single domain antibodiessuch as sdAb (either VL or VH), camelid VHH domains, and multi-specificantibodies formed from antibody fragments. The term “scFv” refers to afusion protein including at least one antibody fragment including avariable region of a light chain and at least one antibody fragmentincluding a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlechain polypeptide, and wherein the scFv retains the specificity of theintact antibody from which it is derived. Unless specified, as usedherein an scFv may have the VL and VH variable regions in either order,e.g., with respect to the N-terminal and C-terminal ends of thepolypeptide, the scFv may comprise VL-linker-VH or may compriseVH-linker-VL.

The term “recombinant polypeptide” refers to a polypeptide which isgenerated using recombinant DNA technology, such as, for example, apolypeptide expressed by a viral vector expression system. The termshould also be construed to mean a polypeptide which has been generatedby the synthesis of a DNA molecule encoding the polypeptide and whichDNA molecule expresses a protein, or an amino acid sequence specifyingthe polypeptide, wherein the DNA or amino acid sequence has beenobtained using recombinant DNA or amino acid sequence technology whichis available and well known in the art.

The term “mutation in an otoferlin gene” refers to a modification in awildtype otoferlin gene that results in the production of an otoferlinprotein having one or more of: a deletion of one or more amino acids,one or more amino acid substitutions, and one or more amino acidinsertions, as compared to the wildtype otoferlin protein, and/orresults in a decrease in the expressed level of the encoded otoferlinprotein in a mammalian cell as compared to the expressed level of theencoded otoferlin protein in a mammalian cell not having the mutation.In some embodiments, a mutation can result in the production of anotoferlin protein having a deletion of one or more amino acids (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 aminoacids). In some embodiments, the mutation can result in a frameshift inthe otoferlin gene. The term “frameshift” is known in the art toencompass any mutation in a coding sequence that results in a shift inthe reading frame of the coding sequence. In some embodiments, aframeshift can result in a nonfunctional protein. In some embodiments, apoint mutation can be a nonsense mutation (i.e., result in a prematurestop codon in an exon of the gene). A nonsense mutation can result inthe production of a truncated protein (as compared to a correspondingwildtype protein) that may or may not be functional. In someembodiments, the mutation can result in the loss (or a decrease in thelevel) of expression of otoferlin mRNA or otoferlin protein or both themRNA and protein. In some embodiments, the mutation can result in theproduction of an altered otoferlin protein having a loss or decrease inone or more biological activities (functions) as compared to a wildtypeotoferlin protein.

In some embodiments, the mutation is an insertion of one or morenucleotides into an otoferlin gene. In some embodiments, the mutation isin a regulatory sequence of the otoferlin gene, i.e., a portion of thegene that is not coding sequence. In some embodiments, a mutation in aregulatory sequence may be in a promoter or enhancer region and preventor reduce the proper transcription of the otoferlin gene.

Modifications can be introduced into a nucleotide sequence by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis.

The term “conservative sequence modifications” refers to amino acidmodifications that do not significantly affect or alter the bindingcharacteristics of the antibody or antibody fragment containing theamino acid sequence. Such conservative modifications include amino acidsubstitutions, additions, and deletions. Modifications can be introducedinto an antibody or antibody fragment of the disclosure by standardtechniques known in the art, such as site-directed mutagenesis andPCR-mediated mutagenesis. Conservative amino acid substitutions are onesin which the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, andhistidine), acidic side chains (e.g., aspartic acid and glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar sidechains (e.g., alanine, valine, leucine, isoleucine, proline,phenylalanine, and methionine), beta-branched side chains (e.g.,threonine, valine, and isoleucine), and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, and histidine).

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of a defined sequence ofamino acids, in accordance with the genetic code. Thus, a gene, cDNA, orRNA encodes a protein if transcription and translation of mRNAcorresponding to that gene, cDNA or RNA produces the protein.

Both the coding strand, the nucleotide sequence of which is identical tothe mRNA sequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription, can bereferred to as encoding the protein product.

The term “homologous” or “identity” refers to the subunit sequenceidentity between two polymeric molecules, e.g., between two nucleic acidmolecules, such as two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous or identical at that position. The homology between twosequences is a direct function of the number of matching or homologouspositions; e.g., if half (e.g., five positions in a polymer ten subunitsin length) of the positions in two sequences are homologous, the twosequences are 50% homologous; if 90% of the positions (e.g., 9 of 10),are matched or homologous, the two sequences are 90% homologous.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and thus encode the same amino acid sequence. Anucleotide sequence that encodes a protein may also include introns.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from outside orproduced outside an organism, cell, tissue or system.

The term “isolated” means altered or removed from the natural state. Forexample, a nucleic acid or a peptide naturally present in a livinganimal is not “isolated,” but the same nucleic acid or peptide partiallyor completely separated from the coexisting materials of its naturalstate is “isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “substantially purified cell” refers to a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cells that have been separated from the cells with which theyare naturally associated in their natural state. In some aspects, thecells are cultured in vitro. In other aspects, the cells are notcultured in vitro.

The term “transfected” or “transformed” or “transduced” refers to aprocess by which exogenous nucleic acid is transferred or introducedinto the host cell. A “transfected” or “transformed” or “transduced”cell is one which has been transfected, transformed or transduced withexogenous nucleic acid. The cell includes the primary subject cell andits progeny.

The term “expression” refers to the transcription and/or translation ofa particular nucleotide sequence driven by a promoter.

As used herein, “transient” refers to expression of a non-integratedtransgene for a period of hours, days or weeks, wherein the period oftime of expression is less than the period of time for expression of thegene if integrated into the genome or contained within a stable plasmidreplicon in the host cell.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals, human). In someembodiments, the subject is a rodent (e.g., a rat or mouse), a rabbit, asheep, a dog, a cat, a horse, a non-human primate, or a human. In someembodiments, the subject has or is at risk of developing non-syndromicdeafness. In some embodiments, the subject has been previouslyidentified as having a mutation in an otoferlin gene. In someembodiments, the subject has been identified as having a mutation in anotoferlin gene and has been diagnosed with non-symptomatic sensorineuralhearing loss. In some embodiments, the subject has been identified ashaving non-symptomatic sensorineural hearing loss.

The term “therapeutic” as used herein means a treatment. A therapeuticeffect is obtained by reduction, suppression, remission, or eradicationof a disease state.

The term “prophylaxis” as used herein means the prevention of, orprotective treatment for, a disease or disease state. “Prevention” inthis context includes reducing the likelihood the subject willexperience the disease.

The term “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition as described herein effective toachieve a particular biological result. In some embodiments, atherapeutically effective amount of a composition can result in anincrease in the expression level of an active otoferlin protein (e.g., awildtype, full-length otoferlin protein or of a variant of an otoferlinprotein that has the desired activity) (e.g., as compared to theexpression level prior to treatment with the composition). In someembodiments, a therapeutically effective amount of a composition canresult in an increase in the expression level of an active otoferlinprotein (e.g., a wildtype, full-length otoferlin protein or activevariant) in a target cell (e.g., a cochlear inner hair cell). In someembodiments, a therapeutically effective amount of a composition canresult in a different cellular localization of an active otoferlinprotein (e.g., a wildtype, full-length otoferlin protein or an activevariant) in a target cell (e.g., a cochlear inner hair cell). In someembodiments, a therapeutically effective amount of a composition canresult in an increase in the expression level of an active otoferlinprotein (e.g., a wildtype, full-length otoferlin protein or activevariant), and/or an increase in one or more activities of an otoferlinprotein in a target cell (e.g., as compared to a reference level, suchas the level(s) in a subject prior to treatment, the level(s) in asubject having a mutation in an otoferlin gene, or the level(s) in asubject or a population of subjects having non-symptomatic sensorineuralhearing loss).

The term “parenteral” administration of a composition includes, e.g.,subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologs, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

In some embodiments of any of the nucleic acids described herein, thenucleic acid is DNA. In some embodiments of any of the nucleic acidsdescribed herein, the nucleic acid is RNA.

In the context of the present disclosure, the following abbreviationsfor the commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

As used herein, “in vitro transcribed RNA” refers to RNA, preferablymRNA, that has been synthesized in vitro. Generally, the in vitrotranscribed RNA is generated from an in vitro transcription vector. Thein vitro transcription vector includes a template that is used togenerate the in vitro transcribed RNA.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein including two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. A polypeptide includes a natural peptide, arecombinant peptide, or a combination thereof.

The term “signal transduction pathway” refers to the biochemicalrelationship between a variety of signal transduction molecules thatplay a role in the transmission of a signal from one portion of a cellto another portion of a cell. The phrase “cell surface receptor”includes molecules and complexes of molecules capable of receiving asignal and transmitting signal across the membrane of a cell.

The term “active otoferlin protein” means a protein encoded by DNA that,if substituted for both wildtype alleles encoding full-length otoferlinprotein in auditory hair cells (e.g., auditory inner hair cells) of whatis otherwise a wildtype mammal, and if expressed in the auditory haircells of that mammal, results in that mammal's having a level of hearingapproximating the normal level of hearing of a similar mammal that isentirely wildtype. Non-limiting examples of active otoferlin proteinsare full-length otoferlin proteins (e.g., any of the full-lengthotoferlin proteins described herein).

For example, an active otoferlin protein can include a sequence of awildtype, full-length otoferlin protein (e.g., a wildtype, human,full-length otoferlin protein) including 1 amino acid substitution toabout 240 amino acid substitutions, 1 amino acid substitution to about235 amino acid substitutions, 1 amino acid substitution to about 230amino acid substitutions, 1 amino acid substitution to about 225 aminoacid substitutions, 1 amino acid substitution to about 220 amino acidsubstitutions, 1 amino acid substitution to about 215 amino acidsubstitutions, 1 amino acid substitution to about 210 amino acidsubstitutions, 1 amino acid substitution to about 205 amino acidsubstitutions, 1 amino acid substitution to about 200 amino acidsubstitutions, 1 amino acid substitution to about 195 amino acidsubstitutions, 1 amino acid substitution to about 190 amino acidsubstitutions, 1 amino acid substitution to about 185 amino acidsubstitutions, 1 amino acid substitution to about 180 amino acidsubstitutions, 1 amino acid substitution to about 175 amino acidsubstitutions, 1 amino acid substitution to about 170 amino acidsubstitutions, 1 amino acid substitution to about 165 amino acidsubstitutions, 1 amino acid substitution to about 160 amino acidsubstitutions, 1 amino acid substitution to about 155 amino acidsubstitutions, 1 amino acid substitution to about 150 amino acidsubstitutions, 1 amino acid substitution to about 145 amino acidsubstitutions, 1 amino acid substitution to about 140 amino acidsubstitutions, 1 amino acid substitution to about 135 amino acidsubstitutions, 1 amino acid substitution to about 130 amino acidsubstitutions, 1 amino acid substitution to about 125 amino acidsubstitutions, 1 amino acid substitution to about 120 amino acidsubstitutions, 1 amino acid substitution to about 115 amino acidsubstitutions, 1 amino acid substitution to about 110 amino acidsubstitutions, 1 amino acid substitution to about 105 amino acidsubstitutions, 1 amino acid substitution to about 100 amino acidsubstitutions, 1 amino acid substitution to about 95 amino acidsubstitutions, 1 amino acid substitution to about 90 amino acidsubstitutions, 1 amino acid substitution to about 85 amino acidsubstitutions, 1 amino acid substitution to about 80 amino acidsubstitutions, 1 amino acid substitution to about 75 amino acidsubstitutions, 1 amino acid substitution to about 70 amino acidsubstitutions, 1 amino acid substitution to about 65 amino acidsubstitutions, 1 amino acid substitution to about 60 amino acidsubstitutions, 1 amino acid substitution to about 55 amino acidsubstitutions, 1 amino acid substitution to about 50 amino acidsubstitutions, 1 amino acid substitution to about 45 amino acidsubstitutions, 1 amino acid substitution to about 40 amino acidsubstitutions, 1 amino acid substitution to about 35 amino acidsubstitutions, 1 amino acid substitution to about 30 amino acidsubstitutions, 1 amino acid substitution to about 25 amino acidsubstitutions, 1 amino acid substitution to about 20 amino acidsubstitutions, 1 amino acid substitution to about 15 amino acidsubstitutions, 1 amino acid substitution to about 10 amino acidsubstitutions, 1 amino acid substitution to about 9 amino acidsubstitutions, 1 amino acid substitution to about 8 amino acidsubstitutions, 1 amino acid substitution to about 7 amino acidsubstitutions, 1 amino acid substitution to about 6 amino acidsubstitutions, 1 amino acid substitution to about 5 amino acidsubstitutions, 1 amino acid substitution to about 4 amino acidsubstitutions, 1 amino acid substitution to about 3 amino acidsubstitutions, between about 2 amino acid substitutions to about 240amino acid substitutions, about 2 amino acid substitutions to about 235amino acid substitutions, about 2 amino acid substitutions to about 230amino acid substitutions, about 2 amino acid substitutions to about 225amino acid substitutions, about 2 amino acid substitutions substitutionto about 220 amino acid substitutions, about 2 amino acid substitutionsto about 215 amino acid substitutions, about 2 amino acid substitutionsto about 210 amino acid substitutions, about 2 amino acid substitutionsto about 205 amino acid substitutions, about 2 amino acid substitutionsto about 200 amino acid substitutions, about 2 amino acid substitutionsto about 195 amino acid substitutions, about 2 amino acid substitutionsto about 190 amino acid substitutions, about 2 amino acid substitutionsto about 185 amino acid substitutions, about 2 amino acid substitutionsto about 180 amino acid substitutions, about 2 amino acid substitutionsto about 175 amino acid substitutions, about 2 amino acid substitutionsto about 170 amino acid substitutions, about 2 amino acid substitutionsto about 165 amino acid substitutions, about 2 amino acid substitutionsto about 160 amino acid substitutions, about 2 amino acid substitutionsto about 155 amino acid substitutions, about 2 amino acid substitutionsto about 150 amino acid substitutions, about 2 amino acid substitutionsto about 145 amino acid substitutions, about 2 amino acid substitutionsto about 140 amino acid substitutions, about 2 amino acid substitutionsto about 135 amino acid substitutions, about 2 amino acid substitutionsto about 130 amino acid substitutions, about 2 amino acid substitutionsto about 125 amino acid substitutions, about 2 amino acid substitutionsto about 120 amino acid substitutions, about 2 amino acid substitutionsto about 115 amino acid substitutions, about 2 amino acid substitutionsto about 110 amino acid substitutions, about 2 amino acid substitutionsto about 105 amino acid substitutions, about 2 amino acid substitutionsto about 100 amino acid substitutions, about 2 amino acid substitutionsto about 95 amino acid substitutions, about 2 amino acid substitutionsto about 90 amino acid substitutions, about 2 amino acid substitutionsto about 85 amino acid substitutions, about 2 amino acid substitutionsto about 80 amino acid substitutions, about 2 amino acid substitutionsto about 75 amino acid substitutions, about 2 amino acid substitutionsto about 70 amino acid substitutions, about 2 amino acid substitutionsto about 65 amino acid substitutions, about 2 amino acid substitutionsto about 60 amino acid substitutions, about 2 amino acid substitutionsto about 55 amino acid substitutions, about 2 amino acid substitutionsto about 50 amino acid substitutions, about 2 amino acid substitutionsto about 45 amino acid substitutions, about 2 amino acid substitutionsto about 40 amino acid substitutions, about 2 amino acid substitutionsto about 35 amino acid substitutions, about 2 amino acid substitutionsto about 30 amino acid substitutions, about 2 amino acid substitutionsto about 25 amino acid substitutions, about 2 amino acid substitutionsto about 20 amino acid substitutions, about 2 amino acid substitutionsto about 15 amino acid substitutions, about 2 amino acid substitutionsto about 10 amino acid substitutions, about 2 amino acid substitutionsto about 9 amino acid substitutions, about 2 amino acid substitutions toabout 8 amino acid substitutions, about 2 amino acid substitutions toabout 7 amino acid substitutions, about 2 amino acid substitutions toabout 6 amino acid substitutions, about 2 amino acid substitutions toabout 5 amino acid substitutions, about 2 amino acid substitutions toabout 4 amino acid substitutions, between about 3 amino acidsubstitutions to about 240 amino acid substitutions, about 3 amino acidsubstitutions to about 235 amino acid substitutions, about 3 amino acidsubstitutions to about 230 amino acid substitutions, about 3 amino acidsubstitutions to about 225 amino acid substitutions, about 3 amino acidsubstitutions substitution to about 220 amino acid substitutions, about3 amino acid substitutions to about 215 amino acid substitutions, about3 amino acid substitutions to about 210 amino acid substitutions, about3 amino acid substitutions to about 205 amino acid substitutions, about3 amino acid substitutions to about 200 amino acid substitutions, about3 amino acid substitutions to about 195 amino acid substitutions, about3 amino acid substitutions to about 190 amino acid substitutions, about3 amino acid substitutions to about 185 amino acid substitutions, about3 amino acid substitutions to about 180 amino acid substitutions, about3 amino acid substitutions to about 175 amino acid substitutions, about3 amino acid substitutions to about 170 amino acid substitutions, about3 amino acid substitutions to about 165 amino acid substitutions, about3 amino acid substitutions to about 160 amino acid substitutions, about3 amino acid substitutions to about 155 amino acid substitutions, about3 amino acid substitutions to about 150 amino acid substitutions, about3 amino acid substitutions to about 145 amino acid substitutions, about3 amino acid substitutions to about 140 amino acid substitutions, about3 amino acid substitutions to about 135 amino acid substitutions, about3 amino acid substitutions to about 130 amino acid substitutions, about3 amino acid substitutions to about 125 amino acid substitutions, about3 amino acid substitutions to about 120 amino acid substitutions, about3 amino acid substitutions to about 115 amino acid substitutions, about3 amino acid substitutions to about 110 amino acid substitutions, about3 amino acid substitutions to about 105 amino acid substitutions, about3 amino acid substitutions to about 100 amino acid substitutions, about3 amino acid substitutions to about 95 amino acid substitutions, about 3amino acid substitutions to about 90 amino acid substitutions, about 3amino acid substitutions to about 85 amino acid substitutions, about 3amino acid substitutions to about 80 amino acid substitutions, about 3amino acid substitutions to about 75 amino acid substitutions, about 3amino acid substitutions to about 70 amino acid substitutions, about 3amino acid substitutions to about 65 amino acid substitutions, about 3amino acid substitutions to about 60 amino acid substitutions, about 3amino acid substitutions to about 55 amino acid substitutions, about 3amino acid substitutions to about 50 amino acid substitutions, about 3amino acid substitutions to about 45 amino acid substitutions, about 3amino acid substitutions to about 40 amino acid substitutions, about 3amino acid substitutions to about 35 amino acid substitutions, about 3amino acid substitutions to about 30 amino acid substitutions, about 3amino acid substitutions to about 25 amino acid substitutions, about 3amino acid substitutions to about 20 amino acid substitutions, about 3amino acid substitutions to about 15 amino acid substitutions, about 3amino acid substitutions to about 10 amino acid substitutions, about 3amino acid substitutions to about 9 amino acid substitutions, about 3amino acid substitutions to about 8 amino acid substitutions, about 3amino acid substitutions to about 7 amino acid substitutions, about 3amino acid substitutions to about 6 amino acid substitutions, about 3amino acid substitutions to about 5 amino acid substitutions, betweenabout 4 amino acid substitutions to about 240 amino acid substitutions,about 4 amino acid substitutions to about 235 amino acid substitutions,about 4 amino acid substitutions to about 230 amino acid substitutions,about 4 amino acid substitutions to about 225 amino acid substitutions,about 4 amino acid substitutions substitution to about 220 amino acidsubstitutions, about 4 amino acid substitutions to about 215 amino acidsubstitutions, about 4 amino acid substitutions to about 210 amino acidsubstitutions, about 4 amino acid substitutions to about 205 amino acidsubstitutions, about 4 amino acid substitutions to about 200 amino acidsubstitutions, about 4 amino acid substitutions to about 195 amino acidsubstitutions, about 4 amino acid substitutions to about 190 amino acidsubstitutions, about 4 amino acid substitutions to about 185 amino acidsubstitutions, about 4 amino acid substitutions to about 180 amino acidsubstitutions, about 4 amino acid substitutions to about 175 amino acidsubstitutions, about 4 amino acid substitutions to about 170 amino acidsubstitutions, about 4 amino acid substitutions to about 165 amino acidsubstitutions, about 4 amino acid substitutions to about 160 amino acidsubstitutions, about 4 amino acid substitutions to about 155 amino acidsubstitutions, about 4 amino acid substitutions to about 150 amino acidsubstitutions, about 4 amino acid substitutions to about 145 amino acidsubstitutions, about 4 amino acid substitutions to about 140 amino acidsubstitutions, about 4 amino acid substitutions to about 135 amino acidsubstitutions, about 4 amino acid substitutions to about 130 amino acidsubstitutions, about 4 amino acid substitutions to about 125 amino acidsubstitutions, about 4 amino acid substitutions to about 120 amino acidsubstitutions, about 4 amino acid substitutions to about 115 amino acidsubstitutions, about 4 amino acid substitutions to about 110 amino acidsubstitutions, about 4 amino acid substitutions to about 105 amino acidsubstitutions, about 4 amino acid substitutions to about 100 amino acidsubstitutions, about 4 amino acid substitutions to about 95 amino acidsubstitutions, about 4 amino acid substitutions to about 90 amino acidsubstitutions, about 4 amino acid substitutions to about 85 amino acidsubstitutions, about 4 amino acid substitutions to about 80 amino acidsubstitutions, about 4 amino acid substitutions to about 75 amino acidsubstitutions, about 4 amino acid substitutions to about 70 amino acidsubstitutions, about 4 amino acid substitutions to about 65 amino acidsubstitutions, about 4 amino acid substitutions to about 60 amino acidsubstitutions, about 4 amino acid substitutions to about 55 amino acidsubstitutions, about 4 amino acid substitutions to about 50 amino acidsubstitutions, about 4 amino acid substitutions to about 45 amino acidsubstitutions, about 4 amino acid substitutions to about 40 amino acidsubstitutions, about 4 amino acid substitutions to about 35 amino acidsubstitutions, about 4 amino acid substitutions to about 30 amino acidsubstitutions, about 4 amino acid substitutions to about 25 amino acidsubstitutions, about 4 amino acid substitutions to about 20 amino acidsubstitutions, about 4 amino acid substitutions to about 15 amino acidsubstitutions, about 4 amino acid substitutions to about 10 amino acidsubstitutions, about 4 amino acid substitutions to about 9 amino acidsubstitutions, about 4 amino acid substitutions to about 8 amino acidsubstitutions, about 4 amino acid substitutions to about 7 amino acidsubstitutions, about 4 amino acid substitutions to about 6 amino acidsubstitutions, between about 5 amino acid substitutions to about 240amino acid substitutions, about 5 amino acid substitutions to about 235amino acid substitutions, about 5 amino acid substitutions to about 230amino acid substitutions, about 5 amino acid substitutions to about 225amino acid substitutions, about 5 amino acid substitutions substitutionto about 220 amino acid substitutions, about 5 amino acid substitutionsto about 215 amino acid substitutions, about 5 amino acid substitutionsto about 210 amino acid substitutions, about 5 amino acid substitutionsto about 205 amino acid substitutions, about 5 amino acid substitutionsto about 200 amino acid substitutions, about 5 amino acid substitutionsto about 195 amino acid substitutions, about 5 amino acid substitutionsto about 190 amino acid substitutions, about 5 amino acid substitutionsto about 185 amino acid substitutions, about 5 amino acid substitutionsto about 180 amino acid substitutions, about 5 amino acid substitutionsto about 175 amino acid substitutions, about 5 amino acid substitutionsto about 170 amino acid substitutions, about 5 amino acid substitutionsto about 165 amino acid substitutions, about 5 amino acid substitutionsto about 160 amino acid substitutions, about 5 amino acid substitutionsto about 155 amino acid substitutions, about 5 amino acid substitutionsto about 150 amino acid substitutions, about 5 amino acid substitutionsto about 145 amino acid substitutions, about 5 amino acid substitutionsto about 140 amino acid substitutions, about 5 amino acid substitutionsto about 135 amino acid substitutions, about 5 amino acid substitutionsto about 130 amino acid substitutions, about 5 amino acid substitutionsto about 125 amino acid substitutions, about 5 amino acid substitutionsto about 120 amino acid substitutions, about 5 amino acid substitutionsto about 115 amino acid substitutions, about 5 amino acid substitutionsto about 110 amino acid substitutions, about 5 amino acid substitutionsto about 105 amino acid substitutions, about 5 amino acid substitutionsto about 100 amino acid substitutions, about 5 amino acid substitutionsto about 95 amino acid substitutions, about 5 amino acid substitutionsto about 90 amino acid substitutions, about 5 amino acid substitutionsto about 85 amino acid substitutions, about 5 amino acid substitutionsto about 80 amino acid substitutions, about 5 amino acid substitutionsto about 75 amino acid substitutions, about 5 amino acid substitutionsto about 70 amino acid substitutions, about 5 amino acid substitutionsto about 65 amino acid substitutions, about 5 amino acid substitutionsto about 60 amino acid substitutions, about 5 amino acid substitutionsto about 55 amino acid substitutions, about 5 amino acid substitutionsto about 50 amino acid substitutions, about 5 amino acid substitutionsto about 45 amino acid substitutions, about 5 amino acid substitutionsto about 40 amino acid substitutions, about 5 amino acid substitutionsto about 35 amino acid substitutions, about 5 amino acid substitutionsto about 30 amino acid substitutions, about 5 amino acid substitutionsto about 25 amino acid substitutions, about 5 amino acid substitutionsto about 20 amino acid substitutions, about 5 amino acid substitutionsto about 15 amino acid substitutions, about 5 amino acid substitutionsto about 10 amino acid substitutions, about 5 amino acid substitutionsto about 9 amino acid substitutions, about 5 amino acid substitutions toabout 8 amino acid substitutions, about 5 amino acid substitutions toabout 7 amino acid substitutions, between about 6 amino acidsubstitutions to about 240 amino acid substitutions, about 6 amino acidsubstitutions to about 235 amino acid substitutions, about 6 amino acidsubstitutions to about 230 amino acid substitutions, about 6 amino acidsubstitutions to about 225 amino acid substitutions, about 6 amino acidsubstitutions substitution to about 220 amino acid substitutions, about6 amino acid substitutions to about 215 amino acid substitutions, about6 amino acid substitutions to about 210 amino acid substitutions, about6 amino acid substitutions to about 205 amino acid substitutions, about6 amino acid substitutions to about 200 amino acid substitutions, about6 amino acid substitutions to about 195 amino acid substitutions, about6 amino acid substitutions to about 190 amino acid substitutions, about6 amino acid substitutions to about 185 amino acid substitutions, about6 amino acid substitutions to about 180 amino acid substitutions, about6 amino acid substitutions to about 175 amino acid substitutions, about6 amino acid substitutions to about 170 amino acid substitutions, about6 amino acid substitutions to about 165 amino acid substitutions, about6 amino acid substitutions to about 160 amino acid substitutions, about6 amino acid substitutions to about 155 amino acid substitutions, about6 amino acid substitutions to about 150 amino acid substitutions, about6 amino acid substitutions to about 145 amino acid substitutions, about6 amino acid substitutions to about 140 amino acid substitutions, about6 amino acid substitutions to about 135 amino acid substitutions, about6 amino acid substitutions to about 130 amino acid substitutions, about6 amino acid substitutions to about 125 amino acid substitutions, about6 amino acid substitutions to about 120 amino acid substitutions, about6 amino acid substitutions to about 115 amino acid substitutions, about6 amino acid substitutions to about 110 amino acid substitutions, about6 amino acid substitutions to about 105 amino acid substitutions, about6 amino acid substitutions to about 100 amino acid substitutions, about6 amino acid substitutions to about 95 amino acid substitutions, about 6amino acid substitutions to about 90 amino acid substitutions, about 6amino acid substitutions to about 85 amino acid substitutions, about 6amino acid substitutions to about 80 amino acid substitutions, about 6amino acid substitutions to about 75 amino acid substitutions, about 6amino acid substitutions to about 70 amino acid substitutions, about 6amino acid substitutions to about 65 amino acid substitutions, about 6amino acid substitutions to about 60 amino acid substitutions, about 6amino acid substitutions to about 55 amino acid substitutions, about 6amino acid substitutions to about 50 amino acid substitutions, about 6amino acid substitutions to about 45 amino acid substitutions, about 6amino acid substitutions to about 40 amino acid substitutions, about 6amino acid substitutions to about 35 amino acid substitutions, about 6amino acid substitutions to about 30 amino acid substitutions, about 6amino acid substitutions to about 25 amino acid substitutions, about 6amino acid substitutions to about 20 amino acid substitutions, about 6amino acid substitutions to about 15 amino acid substitutions, about 6amino acid substitutions to about 10 amino acid substitutions, about 6amino acid substitutions to about 9 amino acid substitutions, about 6amino acid substitutions to about 8 amino acid substitutions, betweenabout 7 amino acid substitutions to about 240 amino acid substitutions,about 7 amino acid substitutions to about 235 amino acid substitutions,about 7 amino acid substitutions to about 230 amino acid substitutions,about 7 amino acid substitutions to about 225 amino acid substitutions,about 7 amino acid substitutions substitution to about 220 amino acidsubstitutions, about 7 amino acid substitutions to about 215 amino acidsubstitutions, about 7 amino acid substitutions to about 210 amino acidsubstitutions, about 7 amino acid substitutions to about 205 amino acidsubstitutions, about 7 amino acid substitutions to about 200 amino acidsubstitutions, about 7 amino acid substitutions to about 195 amino acidsubstitutions, about 7 amino acid substitutions to about 190 amino acidsubstitutions, about 7 amino acid substitutions to about 185 amino acidsubstitutions, about 7 amino acid substitutions to about 180 amino acidsubstitutions, about 7 amino acid substitutions to about 175 amino acidsubstitutions, about 7 amino acid substitutions to about 170 amino acidsubstitutions, about 7 amino acid substitutions to about 165 amino acidsubstitutions, about 7 amino acid substitutions to about 160 amino acidsubstitutions, about 7 amino acid substitutions to about 155 amino acidsubstitutions, about 7 amino acid substitutions to about 150 amino acidsubstitutions, about 7 amino acid substitutions to about 145 amino acidsubstitutions, about 7 amino acid substitutions to about 140 amino acidsubstitutions, about 7 amino acid substitutions to about 135 amino acidsubstitutions, about 7 amino acid substitutions to about 130 amino acidsubstitutions, about 7 amino acid substitutions to about 125 amino acidsubstitutions, about 7 amino acid substitutions to about 120 amino acidsubstitutions, about 7 amino acid substitutions to about 115 amino acidsubstitutions, about 7 amino acid substitutions to about 110 amino acidsubstitutions, about 7 amino acid substitutions to about 105 amino acidsubstitutions, about 7 amino acid substitutions to about 100 amino acidsubstitutions, about 7 amino acid substitutions to about 95 amino acidsubstitutions, about 7 amino acid substitutions to about 90 amino acidsubstitutions, about 7 amino acid substitutions to about 85 amino acidsubstitutions, about 7 amino acid substitutions to about 80 amino acidsubstitutions, about 7 amino acid substitutions to about 75 amino acidsubstitutions, about 7 amino acid substitutions to about 70 amino acidsubstitutions, about 7 amino acid substitutions to about 65 amino acidsubstitutions, about 7 amino acid substitutions to about 60 amino acidsubstitutions, about 7 amino acid substitutions to about 55 amino acidsubstitutions, about 7 amino acid substitutions to about 50 amino acidsubstitutions, about 7 amino acid substitutions to about 45 amino acidsubstitutions, about 7 amino acid substitutions to about 40 amino acidsubstitutions, about 7 amino acid substitutions to about 35 amino acidsubstitutions, about 7 amino acid substitutions to about 30 amino acidsubstitutions, about 7 amino acid substitutions to about 25 amino acidsubstitutions, about 7 amino acid substitutions to about 20 amino acidsubstitutions, about 7 amino acid substitutions to about 15 amino acidsubstitutions, about 7 amino acid substitutions to about 10 amino acidsubstitutions, about 7 amino acid substitutions to about 9 amino acidsubstitutions, between about 8 amino acid substitutions to about 240amino acid substitutions, about 8 amino acid substitutions to about 235amino acid substitutions, about 8 amino acid substitutions to about 230amino acid substitutions, about 8 amino acid substitutions to about 225amino acid substitutions, about 8 amino acid substitutions substitutionto about 220 amino acid substitutions, about 8 amino acid substitutionsto about 215 amino acid substitutions, about 8 amino acid substitutionsto about 210 amino acid substitutions, about 8 amino acid substitutionsto about 205 amino acid substitutions, about 8 amino acid substitutionsto about 200 amino acid substitutions, about 8 amino acid substitutionsto about 195 amino acid substitutions, about 8 amino acid substitutionsto about 190 amino acid substitutions, about 8 amino acid substitutionsto about 185 amino acid substitutions, about 8 amino acid substitutionsto about 180 amino acid substitutions, about 8 amino acid substitutionsto about 175 amino acid substitutions, about 8 amino acid substitutionsto about 170 amino acid substitutions, about 8 amino acid substitutionsto about 165 amino acid substitutions, about 8 amino acid substitutionsto about 160 amino acid substitutions, about 8 amino acid substitutionsto about 155 amino acid substitutions, about 8 amino acid substitutionsto about 150 amino acid substitutions, about 8 amino acid substitutionsto about 145 amino acid substitutions, about 8 amino acid substitutionsto about 140 amino acid substitutions, about 8 amino acid substitutionsto about 135 amino acid substitutions, about 8 amino acid substitutionsto about 130 amino acid substitutions, about 8 amino acid substitutionsto about 125 amino acid substitutions, about 8 amino acid substitutionsto about 120 amino acid substitutions, about 8 amino acid substitutionsto about 115 amino acid substitutions, about 8 amino acid substitutionsto about 110 amino acid substitutions, about 8 amino acid substitutionsto about 105 amino acid substitutions, about 8 amino acid substitutionsto about 100 amino acid substitutions, about 8 amino acid substitutionsto about 95 amino acid substitutions, about 8 amino acid substitutionsto about 90 amino acid substitutions, about 8 amino acid substitutionsto about 85 amino acid substitutions, about 8 amino acid substitutionsto about 80 amino acid substitutions, about 8 amino acid substitutionsto about 75 amino acid substitutions, about 8 amino acid substitutionsto about 70 amino acid substitutions, about 8 amino acid substitutionsto about 65 amino acid substitutions, about 8 amino acid substitutionsto about 60 amino acid substitutions, about 8 amino acid substitutionsto about 55 amino acid substitutions, about 8 amino acid substitutionsto about 50 amino acid substitutions, about 8 amino acid substitutionsto about 45 amino acid substitutions, about 8 amino acid substitutionsto about 40 amino acid substitutions, about 8 amino acid substitutionsto about 35 amino acid substitutions, about 8 amino acid substitutionsto about 30 amino acid substitutions, about 8 amino acid substitutionsto about 25 amino acid substitutions, about 8 amino acid substitutionsto about 20 amino acid substitutions, about 8 amino acid substitutionsto about 15 amino acid substitutions, about 8 amino acid substitutionsto about 10 amino acid substitutions, between about 10 amino acidsubstitutions to about 240 amino acid substitutions, about 10 amino acidsubstitutions to about 235 amino acid substitutions, about 10 amino acidsubstitutions to about 230 amino acid substitutions, about 10 amino acidsubstitutions to about 225 amino acid substitutions, about 10 amino acidsubstitutions substitution to about 220 amino acid substitutions, about10 amino acid substitutions to about 215 amino acid substitutions, about10 amino acid substitutions to about 210 amino acid substitutions, about10 amino acid substitutions to about 205 amino acid substitutions, about10 amino acid substitutions to about 200 amino acid substitutions, about10 amino acid substitutions to about 195 amino acid substitutions, about10 amino acid substitutions to about 190 amino acid substitutions, about10 amino acid substitutions to about 185 amino acid substitutions, about10 amino acid substitutions to about 180 amino acid substitutions, about10 amino acid substitutions to about 175 amino acid substitutions, about10 amino acid substitutions to about 170 amino acid substitutions, about10 amino acid substitutions to about 165 amino acid substitutions, about10 amino acid substitutions to about 160 amino acid substitutions, about10 amino acid substitutions to about 155 amino acid substitutions, about10 amino acid substitutions to about 150 amino acid substitutions, about10 amino acid substitutions to about 145 amino acid substitutions, about10 amino acid substitutions to about 140 amino acid substitutions, about10 amino acid substitutions to about 135 amino acid substitutions, about10 amino acid substitutions to about 130 amino acid substitutions, about10 amino acid substitutions to about 125 amino acid substitutions, about10 amino acid substitutions to about 120 amino acid substitutions, about10 amino acid substitutions to about 115 amino acid substitutions, about10 amino acid substitutions to about 110 amino acid substitutions, about10 amino acid substitutions to about 105 amino acid substitutions, about10 amino acid substitutions to about 100 amino acid substitutions, about10 amino acid substitutions to about 95 amino acid substitutions, about10 amino acid substitutions to about 90 amino acid substitutions, about10 amino acid substitutions to about 85 amino acid substitutions, about10 amino acid substitutions to about 80 amino acid substitutions, about10 amino acid substitutions to about 75 amino acid substitutions, about10 amino acid substitutions to about 70 amino acid substitutions, about10 amino acid substitutions to about 65 amino acid substitutions, about10 amino acid substitutions to about 60 amino acid substitutions, about10 amino acid substitutions to about 55 amino acid substitutions, about10 amino acid substitutions to about 50 amino acid substitutions, about10 amino acid substitutions to about 45 amino acid substitutions, about10 amino acid substitutions to about 40 amino acid substitutions, about10 amino acid substitutions to about 35 amino acid substitutions, about10 amino acid substitutions to about 30 amino acid substitutions, about10 amino acid substitutions to about 25 amino acid substitutions, about10 amino acid substitutions to about 20 amino acid substitutions, about10 amino acid substitutions to about 15 amino acid substitutions,between about 15 amino acid substitutions to about 240 amino acidsubstitutions, about 15 amino acid substitutions to about 235 amino acidsubstitutions, about 15 amino acid substitutions to about 230 amino acidsubstitutions, about 15 amino acid substitutions to about 225 amino acidsubstitutions, about 15 amino acid substitutions substitution to about220 amino acid substitutions, about 15 amino acid substitutions to about215 amino acid substitutions, about 15 amino acid substitutions to about210 amino acid substitutions, about 15 amino acid substitutions to about205 amino acid substitutions, about 15 amino acid substitutions to about200 amino acid substitutions, about 15 amino acid substitutions to about195 amino acid substitutions, about 15 amino acid substitutions to about190 amino acid substitutions, about 15 amino acid substitutions to about185 amino acid substitutions, about 15 amino acid substitutions to about180 amino acid substitutions, about 15 amino acid substitutions to about175 amino acid substitutions, about 15 amino acid substitutions to about170 amino acid substitutions, about 15 amino acid substitutions to about165 amino acid substitutions, about 15 amino acid substitutions to about160 amino acid substitutions, about 15 amino acid substitutions to about155 amino acid substitutions, about 15 amino acid substitutions to about150 amino acid substitutions, about 15 amino acid substitutions to about145 amino acid substitutions, about 15 amino acid substitutions to about140 amino acid substitutions, about 15 amino acid substitutions to about135 amino acid substitutions, about 15 amino acid substitutions to about130 amino acid substitutions, about 15 amino acid substitutions to about125 amino acid substitutions, about 15 amino acid substitutions to about120 amino acid substitutions, about 15 amino acid substitutions to about115 amino acid substitutions, about 15 amino acid substitutions to about110 amino acid substitutions, about 15 amino acid substitutions to about105 amino acid substitutions, about 15 amino acid substitutions to about100 amino acid substitutions, about 15 amino acid substitutions to about95 amino acid substitutions, about 15 amino acid substitutions to about90 amino acid substitutions, about 15 amino acid substitutions to about85 amino acid substitutions, about 15 amino acid substitutions to about80 amino acid substitutions, about 15 amino acid substitutions to about75 amino acid substitutions, about 15 amino acid substitutions to about70 amino acid substitutions, about 15 amino acid substitutions to about65 amino acid substitutions, about 15 amino acid substitutions to about60 amino acid substitutions, about 15 amino acid substitutions to about55 amino acid substitutions, about 15 amino acid substitutions to about50 amino acid substitutions, about 15 amino acid substitutions to about45 amino acid substitutions, about 15 amino acid substitutions to about40 amino acid substitutions, about 15 amino acid substitutions to about35 amino acid substitutions, about 15 amino acid substitutions to about30 amino acid substitutions, about 15 amino acid substitutions to about25 amino acid substitutions, about 15 amino acid substitutions to about20 amino acid substitutions, between about 20 amino acid substitutionsto about 240 amino acid substitutions, about 20 amino acid substitutionsto about 235 amino acid substitutions, about 20 amino acid substitutionsto about 230 amino acid substitutions, about 20 amino acid substitutionsto about 225 amino acid substitutions, about 20 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 20 amino acidsubstitutions to about 215 amino acid substitutions, about 20 amino acidsubstitutions to about 210 amino acid substitutions, about 20 amino acidsubstitutions to about 205 amino acid substitutions, about 20 amino acidsubstitutions to about 200 amino acid substitutions, about 20 amino acidsubstitutions to about 195 amino acid substitutions, about 20 amino acidsubstitutions to about 190 amino acid substitutions, about 20 amino acidsubstitutions to about 185 amino acid substitutions, about 20 amino acidsubstitutions to about 180 amino acid substitutions, about 20 amino acidsubstitutions to about 175 amino acid substitutions, about 20 amino acidsubstitutions to about 170 amino acid substitutions, about 20 amino acidsubstitutions to about 165 amino acid substitutions, about 20 amino acidsubstitutions to about 160 amino acid substitutions, about 20 amino acidsubstitutions to about 155 amino acid substitutions, about 20 amino acidsubstitutions to about 150 amino acid substitutions, about 20 amino acidsubstitutions to about 145 amino acid substitutions, about 20 amino acidsubstitutions to about 140 amino acid substitutions, about 20 amino acidsubstitutions to about 135 amino acid substitutions, about 20 amino acidsubstitutions to about 130 amino acid substitutions, about 20 amino acidsubstitutions to about 125 amino acid substitutions, about 20 amino acidsubstitutions to about 120 amino acid substitutions, about 20 amino acidsubstitutions to about 115 amino acid substitutions, about 20 amino acidsubstitutions to about 110 amino acid substitutions, about 20 amino acidsubstitutions to about 105 amino acid substitutions, about 20 amino acidsubstitutions to about 100 amino acid substitutions, about 20 amino acidsubstitutions to about 95 amino acid substitutions, about 20 amino acidsubstitutions to about 90 amino acid substitutions, about 20 amino acidsubstitutions to about 85 amino acid substitutions, about 20 amino acidsubstitutions to about 80 amino acid substitutions, about 20 amino acidsubstitutions to about 75 amino acid substitutions, about 20 amino acidsubstitutions to about 70 amino acid substitutions, about 20 amino acidsubstitutions to about 65 amino acid substitutions, about 20 amino acidsubstitutions to about 60 amino acid substitutions, about 20 amino acidsubstitutions to about 55 amino acid substitutions, about 20 amino acidsubstitutions to about 50 amino acid substitutions, about 20 amino acidsubstitutions to about 45 amino acid substitutions, about 20 amino acidsubstitutions to about 40 amino acid substitutions, about 20 amino acidsubstitutions to about 35 amino acid substitutions, about 20 amino acidsubstitutions to about 30 amino acid substitutions, about 20 amino acidsubstitutions to about 25 amino acid substitutions, between about 25amino acid substitutions to about 240 amino acid substitutions, about 25amino acid substitutions to about 235 amino acid substitutions, about 25amino acid substitutions to about 230 amino acid substitutions, about 25amino acid substitutions to about 225 amino acid substitutions, about 25amino acid substitutions substitution to about 220 amino acidsubstitutions, about 25 amino acid substitutions to about 215 amino acidsubstitutions, about 25 amino acid substitutions to about 210 amino acidsubstitutions, about 25 amino acid substitutions to about 205 amino acidsubstitutions, about 25 amino acid substitutions to about 200 amino acidsubstitutions, about 10 amino acid substitutions to about 195 amino acidsubstitutions, about 25 amino acid substitutions to about 190 amino acidsubstitutions, about 25 amino acid substitutions to about 185 amino acidsubstitutions, about 25 amino acid substitutions to about 180 amino acidsubstitutions, about 25 amino acid substitutions to about 175 amino acidsubstitutions, about 25 amino acid substitutions to about 170 amino acidsubstitutions, about 25 amino acid substitutions to about 165 amino acidsubstitutions, about 25 amino acid substitutions to about 160 amino acidsubstitutions, about 25 amino acid substitutions to about 155 amino acidsubstitutions, about 25 amino acid substitutions to about 150 amino acidsubstitutions, about 25 amino acid substitutions to about 145 amino acidsubstitutions, about 25 amino acid substitutions to about 140 amino acidsubstitutions, about 25 amino acid substitutions to about 135 amino acidsubstitutions, about 25 amino acid substitutions to about 130 amino acidsubstitutions, about 25 amino acid substitutions to about 125 amino acidsubstitutions, about 25 amino acid substitutions to about 120 amino acidsubstitutions, about 25 amino acid substitutions to about 115 amino acidsubstitutions, about 25 amino acid substitutions to about 110 amino acidsubstitutions, about 25 amino acid substitutions to about 105 amino acidsubstitutions, about 25 amino acid substitutions to about 100 amino acidsubstitutions, about 25 amino acid substitutions to about 95 amino acidsubstitutions, about 25 amino acid substitutions to about 90 amino acidsubstitutions, about 25 amino acid substitutions to about 85 amino acidsubstitutions, about 25 amino acid substitutions to about 80 amino acidsubstitutions, about 25 amino acid substitutions to about 75 amino acidsubstitutions, about 25 amino acid substitutions to about 70 amino acidsubstitutions, about 25 amino acid substitutions to about 65 amino acidsubstitutions, about 25 amino acid substitutions to about 60 amino acidsubstitutions, about 25 amino acid substitutions to about 55 amino acidsubstitutions, about 25 amino acid substitutions to about 50 amino acidsubstitutions, about 25 amino acid substitutions to about 45 amino acidsubstitutions, about 25 amino acid substitutions to about 40 amino acidsubstitutions, about 25 amino acid substitutions to about 35 amino acidsubstitutions, about 25 amino acid substitutions to about 30 amino acidsubstitutions, between about 30 amino acid substitutions to about 240amino acid substitutions, about 30 amino acid substitutions to about 235amino acid substitutions, about 30 amino acid substitutions to about 230amino acid substitutions, about 30 amino acid substitutions to about 225amino acid substitutions, about 30 amino acid substitutions substitutionto about 220 amino acid substitutions, about 30 amino acid substitutionsto about 215 amino acid substitutions, about 30 amino acid substitutionsto about 210 amino acid substitutions, about 30 amino acid substitutionsto about 205 amino acid substitutions, about 30 amino acid substitutionsto about 200 amino acid substitutions, about 30 amino acid substitutionsto about 195 amino acid substitutions, about 30 amino acid substitutionsto about 190 amino acid substitutions, about 30 amino acid substitutionsto about 185 amino acid substitutions, about 30 amino acid substitutionsto about 180 amino acid substitutions, about 30 amino acid substitutionsto about 175 amino acid substitutions, about 30 amino acid substitutionsto about 170 amino acid substitutions, about 30 amino acid substitutionsto about 165 amino acid substitutions, about 30 amino acid substitutionsto about 160 amino acid substitutions, about 30 amino acid substitutionsto about 155 amino acid substitutions, about 30 amino acid substitutionsto about 150 amino acid substitutions, about 30 amino acid substitutionsto about 145 amino acid substitutions, about 30 amino acid substitutionsto about 140 amino acid substitutions, about 30 amino acid substitutionsto about 135 amino acid substitutions, about 30 amino acid substitutionsto about 130 amino acid substitutions, about 30 amino acid substitutionsto about 125 amino acid substitutions, about 30 amino acid substitutionsto about 120 amino acid substitutions, about 30 amino acid substitutionsto about 115 amino acid substitutions, about 30 amino acid substitutionsto about 110 amino acid substitutions, about 30 amino acid substitutionsto about 105 amino acid substitutions, about 30 amino acid substitutionsto about 100 amino acid substitutions, about 30 amino acid substitutionsto about 95 amino acid substitutions, about 30 amino acid substitutionsto about 90 amino acid substitutions, about 30 amino acid substitutionsto about 85 amino acid substitutions, about 30 amino acid substitutionsto about 80 amino acid substitutions, about 30 amino acid substitutionsto about 75 amino acid substitutions, about 30 amino acid substitutionsto about 70 amino acid substitutions, about 30 amino acid substitutionsto about 65 amino acid substitutions, about 30 amino acid substitutionsto about 60 amino acid substitutions, about 30 amino acid substitutionsto about 55 amino acid substitutions, about 30 amino acid substitutionsto about 50 amino acid substitutions, about 30 amino acid substitutionsto about 45 amino acid substitutions, about 30 amino acid substitutionsto about 40 amino acid substitutions, about 30 amino acid substitutionsto about 35 amino acid substitutions, between about 35 amino acidsubstitutions to about 240 amino acid substitutions, about 35 amino acidsubstitutions to about 235 amino acid substitutions, about 35 amino acidsubstitutions to about 230 amino acid substitutions, about 35 amino acidsubstitutions to about 225 amino acid substitutions, about 35 amino acidsubstitutions substitution to about 220 amino acid substitutions, about35 amino acid substitutions to about 215 amino acid substitutions, about35 amino acid substitutions to about 210 amino acid substitutions, about35 amino acid substitutions to about 205 amino acid substitutions, about35 amino acid substitutions to about 200 amino acid substitutions, about35 amino acid substitutions to about 195 amino acid substitutions, about35 amino acid substitutions to about 190 amino acid substitutions, about35 amino acid substitutions to about 185 amino acid substitutions, about35 amino acid substitutions to about 180 amino acid substitutions, about35 amino acid substitutions to about 175 amino acid substitutions, about35 amino acid substitutions to about 170 amino acid substitutions, about35 amino acid substitutions to about 165 amino acid substitutions, about35 amino acid substitutions to about 160 amino acid substitutions, about35 amino acid substitutions to about 155 amino acid substitutions, about35 amino acid substitutions to about 150 amino acid substitutions, about35 amino acid substitutions to about 145 amino acid substitutions, about35 amino acid substitutions to about 140 amino acid substitutions, about35 amino acid substitutions to about 135 amino acid substitutions, about35 amino acid substitutions to about 130 amino acid substitutions, about35 amino acid substitutions to about 125 amino acid substitutions, about35 amino acid substitutions to about 120 amino acid substitutions, about35 amino acid substitutions to about 115 amino acid substitutions, about35 amino acid substitutions to about 110 amino acid substitutions, about35 amino acid substitutions to about 105 amino acid substitutions, about35 amino acid substitutions to about 100 amino acid substitutions, about35 amino acid substitutions to about 95 amino acid substitutions, about35 amino acid substitutions to about 90 amino acid substitutions, about35 amino acid substitutions to about 85 amino acid substitutions, about35 amino acid substitutions to about 80 amino acid substitutions, about35 amino acid substitutions to about 75 amino acid substitutions, about35 amino acid substitutions to about 70 amino acid substitutions, about35 amino acid substitutions to about 65 amino acid substitutions, about35 amino acid substitutions to about 60 amino acid substitutions, about35 amino acid substitutions to about 55 amino acid substitutions, about35 amino acid substitutions to about 50 amino acid substitutions, about35 amino acid substitutions to about 45 amino acid substitutions, about35 amino acid substitutions to about 40 amino acid substitutions,between about 40 amino acid substitutions to about 240 amino acidsubstitutions, about 40 amino acid substitutions to about 235 amino acidsubstitutions, about 40 amino acid substitutions to about 230 amino acidsubstitutions, about 40 amino acid substitutions to about 225 amino acidsubstitutions, about 40 amino acid substitutions substitution to about220 amino acid substitutions, about 40 amino acid substitutions to about215 amino acid substitutions, about 40 amino acid substitutions to about210 amino acid substitutions, about 40 amino acid substitutions to about205 amino acid substitutions, about 40 amino acid substitutions to about200 amino acid substitutions, about 40 amino acid substitutions to about195 amino acid substitutions, about 40 amino acid substitutions to about190 amino acid substitutions, about 40 amino acid substitutions to about185 amino acid substitutions, about 40 amino acid substitutions to about180 amino acid substitutions, about 40 amino acid substitutions to about175 amino acid substitutions, about 40 amino acid substitutions to about170 amino acid substitutions, about 40 amino acid substitutions to about165 amino acid substitutions, about 40 amino acid substitutions to about160 amino acid substitutions, about 40 amino acid substitutions to about155 amino acid substitutions, about 40 amino acid substitutions to about150 amino acid substitutions, about 40 amino acid substitutions to about145 amino acid substitutions, about 40 amino acid substitutions to about140 amino acid substitutions, about 40 amino acid substitutions to about135 amino acid substitutions, about 40 amino acid substitutions to about130 amino acid substitutions, about 40 amino acid substitutions to about125 amino acid substitutions, about 40 amino acid substitutions to about120 amino acid substitutions, about 40 amino acid substitutions to about115 amino acid substitutions, about 40 amino acid substitutions to about110 amino acid substitutions, about 40 amino acid substitutions to about105 amino acid substitutions, about 40 amino acid substitutions to about100 amino acid substitutions, about 40 amino acid substitutions to about95 amino acid substitutions, about 40 amino acid substitutions to about90 amino acid substitutions, about 40 amino acid substitutions to about85 amino acid substitutions, about 40 amino acid substitutions to about80 amino acid substitutions, about 40 amino acid substitutions to about75 amino acid substitutions, about 40 amino acid substitutions to about70 amino acid substitutions, about 40 amino acid substitutions to about65 amino acid substitutions, about 40 amino acid substitutions to about60 amino acid substitutions, about 40 amino acid substitutions to about55 amino acid substitutions, about 40 amino acid substitutions to about50 amino acid substitutions, about 40 amino acid substitutions to about45 amino acid substitutions, between about 45 amino acid substitutionsto about 240 amino acid substitutions, about 45 amino acid substitutionsto about 235 amino acid substitutions, about 45 amino acid substitutionsto about 230 amino acid substitutions, about 45 amino acid substitutionsto about 225 amino acid substitutions, about 45 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 45 amino acidsubstitutions to about 215 amino acid substitutions, about 45 amino acidsubstitutions to about 210 amino acid substitutions, about 45 amino acidsubstitutions to about 205 amino acid substitutions, about 45 amino acidsubstitutions to about 200 amino acid substitutions, about 45 amino acidsubstitutions to about 195 amino acid substitutions, about 45 amino acidsubstitutions to about 190 amino acid substitutions, about 45 amino acidsubstitutions to about 185 amino acid substitutions, about 45 amino acidsubstitutions to about 180 amino acid substitutions, about 45 amino acidsubstitutions to about 175 amino acid substitutions, about 45 amino acidsubstitutions to about 170 amino acid substitutions, about 45 amino acidsubstitutions to about 165 amino acid substitutions, about 45 amino acidsubstitutions to about 160 amino acid substitutions, about 45 amino acidsubstitutions to about 155 amino acid substitutions, about 45 amino acidsubstitutions to about 150 amino acid substitutions, about 45 amino acidsubstitutions to about 145 amino acid substitutions, about 45 amino acidsubstitutions to about 140 amino acid substitutions, about 45 amino acidsubstitutions to about 135 amino acid substitutions, about 45 amino acidsubstitutions to about 130 amino acid substitutions, about 45 amino acidsubstitutions to about 125 amino acid substitutions, about 45 amino acidsubstitutions to about 120 amino acid substitutions, about 45 amino acidsubstitutions to about 115 amino acid substitutions, about 45 amino acidsubstitutions to about 110 amino acid substitutions, about 45 amino acidsubstitutions to about 105 amino acid substitutions, about 45 amino acidsubstitutions to about 100 amino acid substitutions, about 45 amino acidsubstitutions to about 95 amino acid substitutions, about 45 amino acidsubstitutions to about 90 amino acid substitutions, about 45 amino acidsubstitutions to about 85 amino acid substitutions, about 45 amino acidsubstitutions to about 80 amino acid substitutions, about 45 amino acidsubstitutions to about 75 amino acid substitutions, about 45 amino acidsubstitutions to about 70 amino acid substitutions, about 45 amino acidsubstitutions to about 65 amino acid substitutions, about 45 amino acidsubstitutions to about 60 amino acid substitutions, about 45 amino acidsubstitutions to about 55 amino acid substitutions, about 45 amino acidsubstitutions to about 50 amino acid substitutions, between about 50amino acid substitutions to about 240 amino acid substitutions, about 50amino acid substitutions to about 235 amino acid substitutions, about 50amino acid substitutions to about 230 amino acid substitutions, about 50amino acid substitutions to about 225 amino acid substitutions, about 50amino acid substitutions substitution to about 220 amino acidsubstitutions, about 50 amino acid substitutions to about 215 amino acidsubstitutions, about 50 amino acid substitutions to about 210 amino acidsubstitutions, about 50 amino acid substitutions to about 205 amino acidsubstitutions, about 50 amino acid substitutions to about 200 amino acidsubstitutions, about 50 amino acid substitutions to about 195 amino acidsubstitutions, about 50 amino acid substitutions to about 190 amino acidsubstitutions, about 50 amino acid substitutions to about 185 amino acidsubstitutions, about 50 amino acid substitutions to about 180 amino acidsubstitutions, about 50 amino acid substitutions to about 175 amino acidsubstitutions, about 50 amino acid substitutions to about 170 amino acidsubstitutions, about 50 amino acid substitutions to about 165 amino acidsubstitutions, about 50 amino acid substitutions to about 160 amino acidsubstitutions, about 50 amino acid substitutions to about 155 amino acidsubstitutions, about 50 amino acid substitutions to about 150 amino acidsubstitutions, about 50 amino acid substitutions to about 145 amino acidsubstitutions, about 50 amino acid substitutions to about 140 amino acidsubstitutions, about 50 amino acid substitutions to about 135 amino acidsubstitutions, about 50 amino acid substitutions to about 130 amino acidsubstitutions, about 50 amino acid substitutions to about 125 amino acidsubstitutions, about 50 amino acid substitutions to about 120 amino acidsubstitutions, about 50 amino acid substitutions to about 115 amino acidsubstitutions, about 50 amino acid substitutions to about 110 amino acidsubstitutions, about 50 amino acid substitutions to about 105 amino acidsubstitutions, about 50 amino acid substitutions to about 100 amino acidsubstitutions, about 50 amino acid substitutions to about 95 amino acidsubstitutions, about 50 amino acid substitutions to about 90 amino acidsubstitutions, about 50 amino acid substitutions to about 85 amino acidsubstitutions, about 50 amino acid substitutions to about 80 amino acidsubstitutions, about 50 amino acid substitutions to about 75 amino acidsubstitutions, about 50 amino acid substitutions to about 70 amino acidsubstitutions, about 50 amino acid substitutions to about 65 amino acidsubstitutions, about 50 amino acid substitutions to about 60 amino acidsubstitutions, about 50 amino acid substitutions to about 55 amino acidsubstitutions, between about 60 amino acid substitutions to about 240amino acid substitutions, about 60 amino acid substitutions to about 235amino acid substitutions, about 60 amino acid substitutions to about 230amino acid substitutions, about 60 amino acid substitutions to about 225amino acid substitutions, about 60 amino acid substitutions substitutionto about 220 amino acid substitutions, about 60 amino acid substitutionsto about 215 amino acid substitutions, about 60 amino acid substitutionsto about 210 amino acid substitutions, about 60 amino acid substitutionsto about 205 amino acid substitutions, about 60 amino acid substitutionsto about 200 amino acid substitutions, about 60 amino acid substitutionsto about 195 amino acid substitutions, about 60 amino acid substitutionsto about 190 amino acid substitutions, about 60 amino acid substitutionsto about 185 amino acid substitutions, about 60 amino acid substitutionsto about 180 amino acid substitutions, about 60 amino acid substitutionsto about 175 amino acid substitutions, about 60 amino acid substitutionsto about 170 amino acid substitutions, about 60 amino acid substitutionsto about 165 amino acid substitutions, about 60 amino acid substitutionsto about 160 amino acid substitutions, about 60 amino acid substitutionsto about 155 amino acid substitutions, about 60 amino acid substitutionsto about 150 amino acid substitutions, about 60 amino acid substitutionsto about 145 amino acid substitutions, about 60 amino acid substitutionsto about 140 amino acid substitutions, about 60 amino acid substitutionsto about 135 amino acid substitutions, about 60 amino acid substitutionsto about 130 amino acid substitutions, about 60 amino acid substitutionsto about 125 amino acid substitutions, about 60 amino acid substitutionsto about 120 amino acid substitutions, about 60 amino acid substitutionsto about 115 amino acid substitutions, about 60 amino acid substitutionsto about 110 amino acid substitutions, about 60 amino acid substitutionsto about 105 amino acid substitutions, about 60 amino acid substitutionsto about 100 amino acid substitutions, about 60 amino acid substitutionsto about 95 amino acid substitutions, about 60 amino acid substitutionsto about 90 amino acid substitutions, about 60 amino acid substitutionsto about 85 amino acid substitutions, about 60 amino acid substitutionsto about 80 amino acid substitutions, about 60 amino acid substitutionsto about 75 amino acid substitutions, about 60 amino acid substitutionsto about 70 amino acid substitutions, about 60 amino acid substitutionsto about 65 amino acid substitutions, between about 70 amino acidsubstitutions to about 240 amino acid substitutions, about 70 amino acidsubstitutions to about 235 amino acid substitutions, about 70 amino acidsubstitutions to about 230 amino acid substitutions, about 70 amino acidsubstitutions to about 225 amino acid substitutions, about 70 amino acidsubstitutions substitution to about 220 amino acid substitutions, about70 amino acid substitutions to about 215 amino acid substitutions, about70 amino acid substitutions to about 210 amino acid substitutions, about10 amino acid substitutions to about 205 amino acid substitutions, about70 amino acid substitutions to about 200 amino acid substitutions, about70 amino acid substitutions to about 195 amino acid substitutions, about70 amino acid substitutions to about 190 amino acid substitutions, about70 amino acid substitutions to about 185 amino acid substitutions, about70 amino acid substitutions to about 180 amino acid substitutions, about70 amino acid substitutions to about 175 amino acid substitutions, about70 amino acid substitutions to about 170 amino acid substitutions, about70 amino acid substitutions to about 165 amino acid substitutions, about70 amino acid substitutions to about 160 amino acid substitutions, about70 amino acid substitutions to about 155 amino acid substitutions, about70 amino acid substitutions to about 150 amino acid substitutions, about70 amino acid substitutions to about 145 amino acid substitutions, about70 amino acid substitutions to about 140 amino acid substitutions, about70 amino acid substitutions to about 135 amino acid substitutions, about70 amino acid substitutions to about 130 amino acid substitutions, about70 amino acid substitutions to about 125 amino acid substitutions, about70 amino acid substitutions to about 120 amino acid substitutions, about70 amino acid substitutions to about 115 amino acid substitutions, about70 amino acid substitutions to about 110 amino acid substitutions, about70 amino acid substitutions to about 105 amino acid substitutions, about70 amino acid substitutions to about 100 amino acid substitutions, about70 amino acid substitutions to about 95 amino acid substitutions, about70 amino acid substitutions to about 90 amino acid substitutions, about70 amino acid substitutions to about 85 amino acid substitutions, about70 amino acid substitutions to about 80 amino acid substitutions, about70 amino acid substitutions to about 75 amino acid substitutions,between about 80 amino acid substitutions to about 240 amino acidsubstitutions, about 80 amino acid substitutions to about 235 amino acidsubstitutions, about 80 amino acid substitutions to about 230 amino acidsubstitutions, about 80 amino acid substitutions to about 225 amino acidsubstitutions, about 80 amino acid substitutions substitution to about220 amino acid substitutions, about 80 amino acid substitutions to about215 amino acid substitutions, about 80 amino acid substitutions to about210 amino acid substitutions, about 80 amino acid substitutions to about205 amino acid substitutions, about 80 amino acid substitutions to about200 amino acid substitutions, about 80 amino acid substitutions to about195 amino acid substitutions, about 80 amino acid substitutions to about190 amino acid substitutions, about 80 amino acid substitutions to about185 amino acid substitutions, about 80 amino acid substitutions to about180 amino acid substitutions, about 80 amino acid substitutions to about175 amino acid substitutions, about 80 amino acid substitutions to about170 amino acid substitutions, about 80 amino acid substitutions to about165 amino acid substitutions, about 80 amino acid substitutions to about160 amino acid substitutions, about 80 amino acid substitutions to about155 amino acid substitutions, about 80 amino acid substitutions to about150 amino acid substitutions, about 80 amino acid substitutions to about145 amino acid substitutions, about 80 amino acid substitutions to about140 amino acid substitutions, about 80 amino acid substitutions to about135 amino acid substitutions, about 80 amino acid substitutions to about130 amino acid substitutions, about 80 amino acid substitutions to about125 amino acid substitutions, about 80 amino acid substitutions to about120 amino acid substitutions, about 80 amino acid substitutions to about115 amino acid substitutions, about 80 amino acid substitutions to about110 amino acid substitutions, about 80 amino acid substitutions to about105 amino acid substitutions, about 80 amino acid substitutions to about100 amino acid substitutions, about 80 amino acid substitutions to about95 amino acid substitutions, about 80 amino acid substitutions to about90 amino acid substitutions, about 80 amino acid substitutions to about85 amino acid substitutions, between about 90 amino acid substitutionsto about 240 amino acid substitutions, about 90 amino acid substitutionsto about 235 amino acid substitutions, about 90 amino acid substitutionsto about 230 amino acid substitutions, about 90 amino acid substitutionsto about 225 amino acid substitutions, about 90 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 90 amino acidsubstitutions to about 215 amino acid substitutions, about 90 amino acidsubstitutions to about 210 amino acid substitutions, about 90 amino acidsubstitutions to about 205 amino acid substitutions, about 90 amino acidsubstitutions to about 200 amino acid substitutions, about 90 amino acidsubstitutions to about 195 amino acid substitutions, about 90 amino acidsubstitutions to about 190 amino acid substitutions, about 90 amino acidsubstitutions to about 185 amino acid substitutions, about 90 amino acidsubstitutions to about 180 amino acid substitutions, about 90 amino acidsubstitutions to about 175 amino acid substitutions, about 90 amino acidsubstitutions to about 170 amino acid substitutions, about 90 amino acidsubstitutions to about 165 amino acid substitutions, about 90 amino acidsubstitutions to about 160 amino acid substitutions, about 90 amino acidsubstitutions to about 155 amino acid substitutions, about 90 amino acidsubstitutions to about 150 amino acid substitutions, about 90 amino acidsubstitutions to about 145 amino acid substitutions, about 90 amino acidsubstitutions to about 140 amino acid substitutions, about 90 amino acidsubstitutions to about 135 amino acid substitutions, about 90 amino acidsubstitutions to about 130 amino acid substitutions, about 90 amino acidsubstitutions to about 125 amino acid substitutions, about 90 amino acidsubstitutions to about 120 amino acid substitutions, about 90 amino acidsubstitutions to about 115 amino acid substitutions, about 90 amino acidsubstitutions to about 110 amino acid substitutions, about 90 amino acidsubstitutions to about 105 amino acid substitutions, about 90 amino acidsubstitutions to about 100 amino acid substitutions, about 90 amino acidsubstitutions to about 95 amino acid substitutions, between about 100amino acid substitutions to about 240 amino acid substitutions, about100 amino acid substitutions to about 235 amino acid substitutions,about 100 amino acid substitutions to about 230 amino acidsubstitutions, about 100 amino acid substitutions to about 225 aminoacid substitutions, about 100 amino acid substitutions substitution toabout 220 amino acid substitutions, about 100 amino acid substitutionsto about 215 amino acid substitutions, about 100 amino acidsubstitutions to about 210 amino acid substitutions, about 100 aminoacid substitutions to about 205 amino acid substitutions, about 100amino acid substitutions to about 200 amino acid substitutions, about100 amino acid substitutions to about 195 amino acid substitutions,about 100 amino acid substitutions to about 190 amino acidsubstitutions, about 100 amino acid substitutions to about 185 aminoacid substitutions, about 100 amino acid substitutions to about 180amino acid substitutions, about 100 amino acid substitutions to about175 amino acid substitutions, about 100 amino acid substitutions toabout 170 amino acid substitutions, about 100 amino acid substitutionsto about 165 amino acid substitutions, about 100 amino acidsubstitutions to about 160 amino acid substitutions, about 100 aminoacid substitutions to about 155 amino acid substitutions, about 100amino acid substitutions to about 150 amino acid substitutions, about100 amino acid substitutions to about 145 amino acid substitutions,about 100 amino acid substitutions to about 140 amino acidsubstitutions, about 100 amino acid substitutions to about 135 aminoacid substitutions, about 100 amino acid substitutions to about 130amino acid substitutions, about 100 amino acid substitutions to about125 amino acid substitutions, about 100 amino acid substitutions toabout 120 amino acid substitutions, about 100 amino acid substitutionsto about 115 amino acid substitutions, about 100 amino acidsubstitutions to about 110 amino acid substitutions, about 100 aminoacid substitutions to about 105 amino acid substitutions, between about110 amino acid substitutions to about 240 amino acid substitutions,about 110 amino acid substitutions to about 235 amino acidsubstitutions, about 110 amino acid substitutions to about 230 aminoacid substitutions, about 110 amino acid substitutions to about 225amino acid substitutions, about 110 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 110 amino acidsubstitutions to about 215 amino acid substitutions, about 110 aminoacid substitutions to about 210 amino acid substitutions, about 110amino acid substitutions to about 205 amino acid substitutions, about110 amino acid substitutions to about 200 amino acid substitutions,about 110 amino acid substitutions to about 195 amino acidsubstitutions, about 110 amino acid substitutions to about 190 aminoacid substitutions, about 110 amino acid substitutions to about 185amino acid substitutions, about 110 amino acid substitutions to about180 amino acid substitutions, about 110 amino acid substitutions toabout 175 amino acid substitutions, about 110 amino acid substitutionsto about 170 amino acid substitutions, about 110 amino acidsubstitutions to about 165 amino acid substitutions, about 110 aminoacid substitutions to about 160 amino acid substitutions, about 110amino acid substitutions to about 155 amino acid substitutions, about110 amino acid substitutions to about 150 amino acid substitutions,about 110 amino acid substitutions to about 145 amino acidsubstitutions, about 110 amino acid substitutions to about 140 aminoacid substitutions, about 110 amino acid substitutions to about 135amino acid substitutions, about 110 amino acid substitutions to about130 amino acid substitutions, about 110 amino acid substitutions toabout 125 amino acid substitutions, about 110 amino acid substitutionsto about 120 amino acid substitutions, about 110 amino acidsubstitutions to about 115 amino acid substitutions, between about 120amino acid substitutions to about 240 amino acid substitutions, about120 amino acid substitutions to about 235 amino acid substitutions,about 120 amino acid substitutions to about 230 amino acidsubstitutions, about 120 amino acid substitutions to about 225 aminoacid substitutions, about 120 amino acid substitutions substitution toabout 220 amino acid substitutions, about 120 amino acid substitutionsto about 215 amino acid substitutions, about 120 amino acidsubstitutions to about 210 amino acid substitutions, about 120 aminoacid substitutions to about 205 amino acid substitutions, about 120amino acid substitutions to about 200 amino acid substitutions, about120 amino acid substitutions to about 195 amino acid substitutions,about 120 amino acid substitutions to about 190 amino acidsubstitutions, about 120 amino acid substitutions to about 185 aminoacid substitutions, about 120 amino acid substitutions to about 180amino acid substitutions, about 120 amino acid substitutions to about175 amino acid substitutions, about 120 amino acid substitutions toabout 170 amino acid substitutions, about 100 amino acid substitutionsto about 165 amino acid substitutions, about 120 amino acidsubstitutions to about 160 amino acid substitutions, about 120 aminoacid substitutions to about 155 amino acid substitutions, about 120amino acid substitutions to about 150 amino acid substitutions, about120 amino acid substitutions to about 145 amino acid substitutions,about 120 amino acid substitutions to about 140 amino acidsubstitutions, about 120 amino acid substitutions to about 135 aminoacid substitutions, about 120 amino acid substitutions to about 130amino acid substitutions, about 120 amino acid substitutions to about125 amino acid substitutions, between about 130 amino acid substitutionsto about 240 amino acid substitutions, about 130 amino acidsubstitutions to about 235 amino acid substitutions, about 130 aminoacid substitutions to about 230 amino acid substitutions, about 130amino acid substitutions to about 225 amino acid substitutions, about130 amino acid substitutions substitution to about 220 amino acidsubstitutions, about 130 amino acid substitutions to about 215 aminoacid substitutions, about 130 amino acid substitutions to about 210amino acid substitutions, about 130 amino acid substitutions to about205 amino acid substitutions, about 130 amino acid substitutions toabout 200 amino acid substitutions, about 130 amino acid substitutionsto about 195 amino acid substitutions, about 130 amino acidsubstitutions to about 190 amino acid substitutions, about 130 aminoacid substitutions to about 185 amino acid substitutions, about 130amino acid substitutions to about 180 amino acid substitutions, about130 amino acid substitutions to about 175 amino acid substitutions,about 130 amino acid substitutions to about 170 amino acidsubstitutions, about 130 amino acid substitutions to about 165 aminoacid substitutions, about 130 amino acid substitutions to about 160amino acid substitutions, about 130 amino acid substitutions to about155 amino acid substitutions, about 130 amino acid substitutions toabout 150 amino acid substitutions, about 130 amino acid substitutionsto about 145 amino acid substitutions, about 130 amino acidsubstitutions to about 140 amino acid substitutions, about 130 aminoacid substitutions to about 135 amino acid substitutions, between about140 amino acid substitutions to about 240 amino acid substitutions,about 140 amino acid substitutions to about 235 amino acidsubstitutions, about 140 amino acid substitutions to about 230 aminoacid substitutions, about 140 amino acid substitutions to about 225amino acid substitutions, about 140 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 140 amino acidsubstitutions to about 215 amino acid substitutions, about 140 aminoacid substitutions to about 210 amino acid substitutions, about 140amino acid substitutions to about 205 amino acid substitutions, about140 amino acid substitutions to about 200 amino acid substitutions,about 140 amino acid substitutions to about 195 amino acidsubstitutions, about 140 amino acid substitutions to about 190 aminoacid substitutions, about 140 amino acid substitutions to about 185amino acid substitutions, about 140 amino acid substitutions to about180 amino acid substitutions, about 140 amino acid substitutions toabout 175 amino acid substitutions, about 140 amino acid substitutionsto about 170 amino acid substitutions, about 140 amino acidsubstitutions to about 165 amino acid substitutions, about 140 aminoacid substitutions to about 160 amino acid substitutions, about 140amino acid substitutions to about 155 amino acid substitutions, about140 amino acid substitutions to about 150 amino acid substitutions,about 140 amino acid substitutions to about 145 amino acidsubstitutions, between about 150 amino acid substitutions to about 240amino acid substitutions, about 150 amino acid substitutions to about235 amino acid substitutions, about 150 amino acid substitutions toabout 230 amino acid substitutions, about 150 amino acid substitutionsto about 225 amino acid substitutions, about 150 amino acidsubstitutions substitution to about 220 amino acid substitutions, about150 amino acid substitutions to about 215 amino acid substitutions,about 150 amino acid substitutions to about 210 amino acidsubstitutions, about 150 amino acid substitutions to about 205 aminoacid substitutions, about 150 amino acid substitutions to about 200amino acid substitutions, about 150 amino acid substitutions to about195 amino acid substitutions, about 150 amino acid substitutions toabout 190 amino acid substitutions, about 150 amino acid substitutionsto about 185 amino acid substitutions, about 150 amino acidsubstitutions to about 180 amino acid substitutions, about 150 aminoacid substitutions to about 175 amino acid substitutions, about 150amino acid substitutions to about 170 amino acid substitutions, about150 amino acid substitutions to about 165 amino acid substitutions,about 150 amino acid substitutions to about 160 amino acidsubstitutions, or about 150 amino acid substitutions to about 155 aminoacid substitutions, between about 160 amino acid substitutions to about240 amino acid substitutions, about 160 amino acid substitutions toabout 235 amino acid substitutions, about 160 amino acid substitutionsto about 230 amino acid substitutions, about 160 amino acidsubstitutions to about 225 amino acid substitutions, about 160 aminoacid substitutions substitution to about 220 amino acid substitutions,about 160 amino acid substitutions to about 215 amino acidsubstitutions, about 160 amino acid substitutions to about 210 aminoacid substitutions, about 160 amino acid substitutions to about 205amino acid substitutions, about 160 amino acid substitutions to about200 amino acid substitutions, about 160 amino acid substitutions toabout 195 amino acid substitutions, about 160 amino acid substitutionsto about 190 amino acid substitutions, about 160 amino acidsubstitutions to about 185 amino acid substitutions, about 160 aminoacid substitutions to about 180 amino acid substitutions, about 160amino acid substitutions to about 175 amino acid substitutions, about160 amino acid substitutions to about 170 amino acid substitutions,about 160 amino acid substitutions to about 165 amino acidsubstitutions, between about 170 amino acid substitutions to about 240amino acid substitutions, about 170 amino acid substitutions to about235 amino acid substitutions, about 170 amino acid substitutions toabout 230 amino acid substitutions, about 170 amino acid substitutionsto about 225 amino acid substitutions, about 170 amino acidsubstitutions substitution to about 220 amino acid substitutions, about170 amino acid substitutions to about 215 amino acid substitutions,about 170 amino acid substitutions to about 210 amino acidsubstitutions, about 170 amino acid substitutions to about 205 aminoacid substitutions, about 170 amino acid substitutions to about 200amino acid substitutions, about 170 amino acid substitutions to about195 amino acid substitutions, about 170 amino acid substitutions toabout 190 amino acid substitutions, about 170 amino acid substitutionsto about 185 amino acid substitutions, about 170 amino acidsubstitutions to about 180 amino acid substitutions, about 170 aminoacid substitutions to about 175 amino acid substitutions, between about180 amino acid substitutions to about 240 amino acid substitutions,about 180 amino acid substitutions to about 235 amino acidsubstitutions, about 180 amino acid substitutions to about 230 aminoacid substitutions, about 180 amino acid substitutions to about 225amino acid substitutions, about 180 amino acid substitutionssubstitution to about 220 amino acid substitutions, about 180 amino acidsubstitutions to about 215 amino acid substitutions, about 180 aminoacid substitutions to about 210 amino acid substitutions, about 180amino acid substitutions to about 205 amino acid substitutions, about180 amino acid substitutions to about 200 amino acid substitutions,about 180 amino acid substitutions to about 195 amino acidsubstitutions, about 180 amino acid substitutions to about 190 aminoacid substitutions, about 180 amino acid substitutions to about 185amino acid substitutions, between about 190 amino acid substitutions toabout 240 amino acid substitutions, about 190 amino acid substitutionsto about 235 amino acid substitutions, about 190 amino acidsubstitutions to about 230 amino acid substitutions, about 190 aminoacid substitutions to about 225 amino acid substitutions, about 190amino acid substitutions substitution to about 220 amino acidsubstitutions, about 190 amino acid substitutions to about 215 aminoacid substitutions, about 190 amino acid substitutions to about 210amino acid substitutions, about 190 amino acid substitutions to about205 amino acid substitutions, about 190 amino acid substitutions toabout 200 amino acid substitutions, about 190 amino acid substitutionsto about 195 amino acid substitutions, between about 200 amino acidsubstitutions to about 240 amino acid substitutions, about 200 aminoacid substitutions to about 235 amino acid substitutions, about 200amino acid substitutions to about 230 amino acid substitutions, about200 amino acid substitutions to about 225 amino acid substitutions,about 200 amino acid substitutions substitution to about 220 amino acidsubstitutions, about 200 amino acid substitutions to about 215 aminoacid substitutions, about 200 amino acid substitutions to about 210amino acid substitutions, about 200 amino acid substitutions to about205 amino acid substitutions, between about 205 amino acid substitutionsto about 240 amino acid substitutions, about 205 amino acidsubstitutions to about 235 amino acid substitutions, about 205 aminoacid substitutions to about 230 amino acid substitutions, about 205amino acid substitutions to about 225 amino acid substitutions, about205 amino acid substitutions substitution to about 220 amino acidsubstitutions, about 205 amino acid substitutions to about 215 aminoacid substitutions, about 205 amino acid substitutions to about 210amino acid substitutions, between about 210 amino acid substitutions toabout 240 amino acid substitutions, about 210 amino acid substitutionsto about 235 amino acid substitutions, about 210 amino acidsubstitutions to about 230 amino acid substitutions, about 210 aminoacid substitutions to about 225 amino acid substitutions, about 210amino acid substitutions substitution to about 220 amino acidsubstitutions, about 210 amino acid substitutions to about 215 aminoacid substitutions, between about 215 amino acid substitutions to about240 amino acid substitutions, about 215 amino acid substitutions toabout 235 amino acid substitutions, about 215 amino acid substitutionsto about 230 amino acid substitutions, about 215 amino acidsubstitutions to about 225 amino acid substitutions, about 215 aminoacid substitutions substitution to about 220 amino acid substitutions,between about 220 amino acid substitutions to about 240 amino acidsubstitutions, about 220 amino acid substitutions to about 235 aminoacid substitutions, about 220 amino acid substitutions to about 230amino acid substitutions, about 220 amino acid substitutions to about225 amino acid substitutions, between about 225 amino acid substitutionsto about 240 amino acid substitutions, about 225 amino acidsubstitutions to about 235 amino acid substitutions, about 225 aminoacid substitutions to about 230 amino acid substitutions, between about230 amino acid substitutions to about 240 amino acid substitutions,about 230 amino acid substitutions to about 235 amino acidsubstitutions. One skilled in the art would appreciate that amino acidsthat are not conserved between wildtype otoferlin proteins fromdifferent species can be mutated without losing activity, while thoseamino acids that are conserved between wildtype otoferlin proteins fromdifferent species should not be mutated as they are more likely (thanamino acids that are not conserved between different species) to beinvolved in activity.

An active otoferlin protein can include, e.g., a sequence of a wildtype,full-length otoferlin protein (e.g., a wildtype, human, full-lengthotoferlin protein) that has 1 amino acid to about 200 amino acids, 1amino acid to about 195 amino acids, 1 amino acid to about 190 aminoacids, 1 amino acid to about 185 amino acids, 1 amino acid to about 180amino acids, 1 amino acid to about 175 amino acids, 1 amino acid toabout 170 amino acids, 1 amino acid to about 165 amino acids, 1 aminoacid to about 160 amino acids, 1 amino acid to about 155 amino acids, 1amino acid to about 150 amino acids, 1 amino acid to about 145 aminoacids, 1 amino acid to about 140 amino acids, 1 amino acid to about 135amino acids, 1 amino acid to about 130 amino acids, 1 amino acid toabout 125 amino acids, 1 amino acid to about 120 amino acids, 1 aminoacid to about 115 amino acids, 1 amino acid to about 110 amino acids, 1amino acid to about 105 amino acids, 1 amino acid to about 100 aminoacids, 1 amino acid to about 95 amino acids, 1 amino acid to about 90amino acids, 1 amino acid to about 85 amino acids, 1 amino acid to about80 amino acids, 1 amino acid to about 75 amino acids, 1 amino acid toabout 70 amino acids, 1 amino acid to about 65 amino acids, 1 amino acidto about 60 amino acids, 1 amino acid to about 55 amino acids, 1 aminoacid to about 50 amino acids, 1 amino acid to about 45 amino acids, 1amino acid to about 40 amino acids, 1 amino acid to about 35 aminoacids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about15 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid toabout 9 amino acids, 1 amino acid to about 8 amino acids, 1 amino acidto about 7 amino acids, 1 amino acid to about 6 amino acids, 1 aminoacid to about 5 amino acids, 1 amino acid to about 4 amino acids, 1amino acid to about 3 amino acids, about 2 amino acids to about 200amino acids, about 2 amino acids to about 195 amino acids, about 2 aminoacids to about 190 amino acids, about 2 amino acids to about 185 aminoacids, about 2 amino acids to about 180 amino acids, about 2 amino acidsto about 175 amino acids, about 2 amino acids to about 170 amino acids,about 2 amino acids to about 165 amino acids, about 2 amino acids toabout 160 amino acids, about 2 amino acids to about 155 amino acids,about 2 amino acids to about 150 amino acids, about 2 amino acids toabout 145 amino acids, about 2 amino acids to about 140 amino acids,about 2 amino acids to about 135 amino acids, about 2 amino acids toabout 130 amino acids, about 2 amino acids to about 125 amino acids,about 2 amino acids to about 120 amino acids, about 2 amino acids toabout 115 amino acids, about 2 amino acids to about 110 amino acids,about 2 amino acids to about 105 amino acids, about 2 amino acids toabout 100 amino acids, about 2 amino acids to about 95 amino acids,about 2 amino acids to about 90 amino acids, about 2 amino acids toabout 85 amino acids, about 2 amino acids to about 80 amino acids, about2 amino acids to about 75 amino acids, about 2 amino acids to about 70amino acids, about 2 amino acids to about 65 amino acids, about 2 aminoacids to about 60 amino acids, about 2 amino acids to about 55 aminoacids, about 2 amino acids to about 50 amino acids, about 2 amino acidsto about 45 amino acids, about 2 amino acids to about 40 amino acids,about 2 amino acids to about 35 amino acids, about 2 amino acids toabout 30 amino acids, about 2 amino acids to about 25 amino acids, about2 amino acids to about 20 amino acids, about 2 amino acids to about 15amino acids, about 2 amino acids to about 10 amino acids, about 2 aminoacids to about 9 amino acids, about 2 amino acids to about 8 aminoacids, about 2 amino acids to about 7 amino acids, about 2 amino acidsto about 6 amino acids, about 2 amino acids to about 5 amino acids,about 2 amino acids to about 4 amino acids, about 3 amino acids to about200 amino acids, about 3 amino acids to about 195 amino acids, about 3amino acids to about 190 amino acids, about 3 amino acids to about 185amino acids, about 3 amino acids to about 180 amino acids, about 3 aminoacids to about 175 amino acids, about 3 amino acids to about 170 aminoacids, about 3 amino acids to about 165 amino acids, about 3 amino acidsto about 160 amino acids, about 3 amino acids to about 155 amino acids,about 3 amino acids to about 150 amino acids, about 3 amino acids toabout 145 amino acids, about 3 amino acids to about 140 amino acids,about 3 amino acids to about 135 amino acids, about 3 amino acids toabout 130 amino acids, about 3 amino acids to about 125 amino acids,about 3 amino acids to about 120 amino acids, about 3 amino acids toabout 115 amino acids, about 3 amino acids to about 110 amino acids,about 3 amino acids to about 105 amino acids, about 3 amino acids toabout 100 amino acids, about 3 amino acids to about 95 amino acids,about 3 amino acids to about 90 amino acids, about 3 amino acids toabout 85 amino acids, about 3 amino acids to about 80 amino acids, about3 amino acids to about 75 amino acids, about 3 amino acids to about 70amino acids, about 3 amino acids to about 65 amino acids, about 3 aminoacids to about 60 amino acids, about 3 amino acids to about 55 aminoacids, about 3 amino acids to about 50 amino acids, about 3 amino acidsto about 45 amino acids, about 3 amino acids to about 40 amino acids,about 3 amino acids to about 35 amino acids, about 3 amino acids toabout 30 amino acids, about 3 amino acids to about 25 amino acids, about3 amino acids to about 20 amino acids, about 3 amino acids to about 15amino acids, about 3 amino acids to about 10 amino acids, about 3 aminoacids to about 9 amino acids, about 3 amino acids to about 8 aminoacids, about 3 amino acids to about 7 amino acids, about 3 amino acidsto about 6 amino acids, about 3 amino acids to about 5 amino acids,about 4 amino acids to about 200 amino acids, about 4 amino acids toabout 195 amino acids, about 4 amino acids to about 190 amino acids,about 4 amino acids to about 185 amino acids, about 4 amino acids toabout 180 amino acids, about 4 amino acids to about 175 amino acids,about 4 amino acids to about 170 amino acids, about 4 amino acids toabout 165 amino acids, about 4 amino acids to about 160 amino acids,about 4 amino acids to about 155 amino acids, about 4 amino acids toabout 150 amino acids, about 4 amino acids to about 145 amino acids,about 4 amino acids to about 140 amino acids, about 4 amino acids toabout 135 amino acids, about 4 amino acids to about 130 amino acids,about 4 amino acids to about 125 amino acids, about 4 amino acids toabout 120 amino acids, about 4 amino acids to about 115 amino acids,about 4 amino acids to about 110 amino acids, about 4 amino acids toabout 105 amino acids, about 4 amino acids to about 100 amino acids,about 4 amino acids to about 95 amino acids, about 4 amino acids toabout 90 amino acids, about 4 amino acids to about 85 amino acids, about4 amino acids to about 80 amino acids, about 4 amino acids to about 75amino acids, about 4 amino acids to about 70 amino acids, about 4 aminoacids to about 65 amino acids, about 4 amino acids to about 60 aminoacids, about 4 amino acids to about 55 amino acids, about 4 amino acidsto about 50 amino acids, about 4 amino acids to about 45 amino acids,about 4 amino acids to about 40 amino acids, about 4 amino acids toabout 35 amino acids, about 4 amino acids to about 30 amino acids, about4 amino acids to about 25 amino acids, about 4 amino acids to about 20amino acids, about 4 amino acids to about 15 amino acids, about 4 aminoacids to about 10 amino acids, about 4 amino acids to about 9 aminoacids, about 4 amino acids to about 8 amino acids, about 4 amino acidsto about 7 amino acids, about 4 amino acids to about 6 amino acids,about 5 amino acids to about 200 amino acids, about 5 amino acids toabout 195 amino acids, about 5 amino acids to about 190 amino acids,about 5 amino acids to about 185 amino acids, about 5 amino acids toabout 180 amino acids, about 5 amino acids to about 175 amino acids,about 5 amino acids to about 170 amino acids, about 5 amino acids toabout 165 amino acids, about 5 amino acids to about 160 amino acids,about 5 amino acids to about 155 amino acids, about 5 amino acids toabout 150 amino acids, about 5 amino acids to about 145 amino acids,about 5 amino acids to about 140 amino acids, about 5 amino acids toabout 135 amino acids, about 5 amino acids to about 130 amino acids,about 5 amino acids to about 125 amino acids, about 5 amino acids toabout 120 amino acids, about 5 amino acids to about 115 amino acids,about 5 amino acids to about 110 amino acids, about 5 amino acids toabout 105 amino acids, about 5 amino acids to about 100 amino acids,about 5 amino acids to about 95 amino acids, about 5 amino acids toabout 90 amino acids, about 5 amino acids to about 85 amino acids, about5 amino acids to about 80 amino acids, about 5 amino acids to about 75amino acids, about 5 amino acids to about 70 amino acids, about 5 aminoacids to about 65 amino acids, about 5 amino acids to about 60 aminoacids, about 5 amino acids to about 55 amino acids, about 5 amino acidsto about 50 amino acids, about 5 amino acids to about 45 amino acids,about 5 amino acids to about 40 amino acids, about 5 amino acids toabout 35 amino acids, about 5 amino acids to about 30 amino acids, about5 amino acids to about 25 amino acids, about 5 amino acids to about 20amino acids, about 5 amino acids to about 15 amino acids, about 5 aminoacids to about 10 amino acids, about 5 amino acids to about 9 aminoacids, about 5 amino acids to about 8 amino acids, about 5 amino acidsto about 7 amino acids, about 6 amino acids to about 200 amino acids,about 6 amino acids to about 195 amino acids, about 6 amino acids toabout 190 amino acids, about 6 amino acids to about 185 amino acids,about 6 amino acids to about 180 amino acids, about 6 amino acids toabout 175 amino acids, about 6 amino acids to about 170 amino acids,about 6 amino acids to about 165 amino acids, about 6 amino acids toabout 160 amino acids, about 6 amino acids to about 155 amino acids,about 6 amino acids to about 150 amino acids, about 6 amino acids toabout 145 amino acids, about 6 amino acids to about 140 amino acids,about 6 amino acids to about 135 amino acids, about 6 amino acids toabout 130 amino acids, about 6 amino acids to about 125 amino acids,about 6 amino acids to about 120 amino acids, about 6 amino acids toabout 115 amino acids, about 6 amino acids to about 110 amino acids,about 6 amino acids to about 105 amino acids, about 6 amino acids toabout 100 amino acids, about 6 amino acids to about 95 amino acids,about 6 amino acids to about 90 amino acids, about 6 amino acids toabout 85 amino acids, about 6 amino acids to about 80 amino acids, about6 amino acids to about 75 amino acids, about 6 amino acids to about 70amino acids, about 6 amino acids to about 65 amino acids, about 6 aminoacids to about 60 amino acids, about 6 amino acids to about 55 aminoacids, about 6 amino acids to about 50 amino acids, about 6 amino acidsto about 45 amino acids, about 6 amino acids to about 40 amino acids,about 6 amino acids to about 35 amino acids, about 6 amino acids toabout 30 amino acids, about 6 amino acids to about 25 amino acids, about6 amino acids to about 20 amino acids, about 6 amino acids to about 15amino acids, about 6 amino acids to about 10 amino acids, about 6 aminoacids to about 9 amino acids, about 6 amino acids to about 8 aminoacids, about 7 amino acids to about 200 amino acids, about 7 amino acidsto about 195 amino acids, about 7 amino acids to about 190 amino acids,about 7 amino acids to about 185 amino acids, about 7 amino acids toabout 180 amino acids, about 7 amino acids to about 175 amino acids,about 7 amino acids to about 170 amino acids, about 7 amino acids toabout 165 amino acids, about 7 amino acids to about 160 amino acids,about 7 amino acids to about 155 amino acids, about 7 amino acids toabout 150 amino acids, about 7 amino acids to about 145 amino acids,about 7 amino acids to about 140 amino acids, about 7 amino acids toabout 135 amino acids, about 7 amino acids to about 130 amino acids,about 7 amino acids to about 125 amino acids, about 7 amino acids toabout 120 amino acids, about 7 amino acids to about 115 amino acids,about 7 amino acids to about 110 amino acids, about 7 amino acids toabout 105 amino acids, about 7 amino acids to about 100 amino acids,about 7 amino acids to about 95 amino acids, about 7 amino acids toabout 90 amino acids, about 7 amino acids to about 85 amino acids, about7 amino acids to about 80 amino acids, about 7 amino acids to about 75amino acids, about 7 amino acids to about 70 amino acids, about 7 aminoacids to about 65 amino acids, about 7 amino acids to about 60 aminoacids, about 7 amino acids to about 55 amino acids, about 7 amino acidsto about 50 amino acids, about 7 amino acids to about 45 amino acids,about 7 amino acids to about 40 amino acids, about 7 amino acids toabout 35 amino acids, about 7 amino acids to about 30 amino acids, about7 amino acids to about 25 amino acids, about 7 amino acids to about 20amino acids, about 7 amino acids to about 15 amino acids, about 7 aminoacids to about 10 amino acids, about 7 amino acids to about 9 aminoacids, about 8 amino acids to about 200 amino acids, about 8 amino acidsto about 195 amino acids, about 8 amino acids to about 190 amino acids,about 8 amino acids to about 185 amino acids, about 8 amino acids toabout 180 amino acids, about 8 amino acids to about 175 amino acids,about 8 amino acids to about 170 amino acids, about 8 amino acids toabout 165 amino acids, about 8 amino acids to about 160 amino acids,about 8 amino acids to about 155 amino acids, about 8 amino acids toabout 150 amino acids, about 8 amino acids to about 145 amino acids,about 8 amino acids to about 140 amino acids, about 8 amino acids toabout 135 amino acids, about 8 amino acids to about 130 amino acids,about 8 amino acids to about 125 amino acids, about 8 amino acids toabout 120 amino acids, about 8 amino acids to about 115 amino acids,about 8 amino acids to about 110 amino acids, about 8 amino acids toabout 105 amino acids, about 8 amino acids to about 100 amino acids,about 8 amino acids to about 95 amino acids, about 8 amino acids toabout 90 amino acids, about 8 amino acids to about 85 amino acids, about8 amino acids to about 80 amino acids, about 8 amino acids to about 75amino acids, about 8 amino acids to about 70 amino acids, about 8 aminoacids to about 65 amino acids, about 8 amino acids to about 60 aminoacids, about 8 amino acids to about 55 amino acids, about 8 amino acidsto about 50 amino acids, about 8 amino acids to about 45 amino acids,about 8 amino acids to about 40 amino acids, about 8 amino acids toabout 35 amino acids, about 8 amino acids to about 30 amino acids, about8 amino acids to about 25 amino acids, about 8 amino acids to about 20amino acids, about 8 amino acids to about 15 amino acids, about 8 aminoacids to about 10 amino acids, about 10 amino acids to about 200 aminoacids, about 10 amino acids to about 195 amino acids, about 10 aminoacids to about 190 amino acids, about 10 amino acids to about 185 aminoacids, about 10 amino acids to about 180 amino acids, about 10 aminoacids to about 175 amino acids, about 10 amino acids to about 170 aminoacids, about 10 amino acids to about 165 amino acids, about 10 aminoacids to about 160 amino acids, about 10 amino acids to about 155 aminoacids, about 10 amino acids to about 150 amino acids, about 10 aminoacids to about 145 amino acids, about 10 amino acids to about 140 aminoacids, about 10 amino acids to about 135 amino acids, about 10 aminoacids to about 130 amino acids, about 10 amino acids to about 125 aminoacids, about 10 amino acids to about 120 amino acids, about 10 aminoacids to about 115 amino acids, about 10 amino acids to about 110 aminoacids, about 10 amino acids to about 105 amino acids, about 10 aminoacids to about 100 amino acids, about 10 amino acids to about 95 aminoacids, about 10 amino acids to about 90 amino acids, about 10 aminoacids to about 85 amino acids, about 10 amino acids to about 80 aminoacids, about 10 amino acids to about 75 amino acids, about 10 aminoacids to about 70 amino acids, about 10 amino acids to about 65 aminoacids, about 10 amino acids to about 60 amino acids, about 10 aminoacids to about 55 amino acids, about 10 amino acids to about 50 aminoacids, about 10 amino acids to about 45 amino acids, about 10 aminoacids to about 40 amino acids, about 10 amino acids to about 35 aminoacids, about 10 amino acids to about 30 amino acids, about 10 aminoacids to about 25 amino acids, about 10 amino acids to about 20 aminoacids, about 10 amino acids to about 15 amino acids, about 15 aminoacids to about 200 amino acids, about 15 amino acids to about 195 aminoacids, about 15 amino acids to about 190 amino acids, about 15 aminoacids to about 185 amino acids, about 15 amino acids to about 180 aminoacids, about 15 amino acids to about 175 amino acids, about 15 aminoacids to about 170 amino acids, about 15 amino acids to about 165 aminoacids, about 15 amino acids to about 160 amino acids, about 15 aminoacids to about 155 amino acids, about 15 amino acids to about 150 aminoacids, about 15 amino acids to about 145 amino acids, about 15 aminoacids to about 140 amino acids, about 15 amino acids to about 135 aminoacids, about 15 amino acids to about 130 amino acids, about 15 aminoacids to about 125 amino acids, about 15 amino acids to about 120 aminoacids, about 15 amino acids to about 115 amino acids, about 15 aminoacids to about 110 amino acids, about 15 amino acids to about 105 aminoacids, about 15 amino acids to about 100 amino acids, about 15 aminoacids to about 95 amino acids, about 15 amino acids to about 90 aminoacids, about 15 amino acids to about 85 amino acids, about 15 aminoacids to about 80 amino acids, about 15 amino acids to about 75 aminoacids, about 15 amino acids to about 70 amino acids, about 15 aminoacids to about 65 amino acids, about 15 amino acids to about 60 aminoacids, about 15 amino acids to about 55 amino acids, about 15 aminoacids to about 50 amino acids, about 15 amino acids to about 45 aminoacids, about 15 amino acids to about 40 amino acids, about 15 aminoacids to about 35 amino acids, about 15 amino acids to about 30 aminoacids, about 15 amino acids to about 25 amino acids, about 15 aminoacids to about 20 amino acids, about 20 amino acids to about 200 aminoacids, about 20 amino acids to about 195 amino acids, about 20 aminoacids to about 190 amino acids, about 20 amino acids to about 185 aminoacids, about 20 amino acids to about 180 amino acids, about 20 aminoacids to about 175 amino acids, about 20 amino acids to about 170 aminoacids, about 20 amino acids to about 165 amino acids, about 20 aminoacids to about 160 amino acids, about 20 amino acids to about 155 aminoacids, about 20 amino acids to about 150 amino acids, about 20 aminoacids to about 145 amino acids, about 20 amino acids to about 140 aminoacids, about 20 amino acids to about 135 amino acids, about 20 aminoacids to about 130 amino acids, about 20 amino acids to about 125 aminoacids, about 20 amino acids to about 120 amino acids, about 20 aminoacids to about 115 amino acids, about 20 amino acids to about 110 aminoacids, about 20 amino acids to about 105 amino acids, about 20 aminoacids to about 100 amino acids, about 20 amino acids to about 95 aminoacids, about 20 amino acids to about 90 amino acids, about 20 aminoacids to about 85 amino acids, about 20 amino acids to about 80 aminoacids, about 20 amino acids to about 75 amino acids, about 20 aminoacids to about 70 amino acids, about 20 amino acids to about 65 aminoacids, about 20 amino acids to about 60 amino acids, about 20 aminoacids to about 55 amino acids, about 20 amino acids to about 50 aminoacids, about 20 amino acids to about 45 amino acids, about 20 aminoacids to about 40 amino acids, about 20 amino acids to about 35 aminoacids, about 20 amino acids to about 30 amino acids, about 20 aminoacids to about 25 amino acids, about 25 amino acids to about 200 aminoacids, about 25 amino acids to about 195 amino acids, about 25 aminoacids to about 190 amino acids, about 25 amino acids to about 185 aminoacids, about 25 amino acids to about 180 amino acids, about 25 aminoacids to about 175 amino acids, about 25 amino acids to about 170 aminoacids, about 25 amino acids to about 165 amino acids, about 25 aminoacids to about 160 amino acids, about 25 amino acids to about 155 aminoacids, about 25 amino acids to about 150 amino acids, about 25 aminoacids to about 145 amino acids, about 25 amino acids to about 140 aminoacids, about 25 amino acids to about 135 amino acids, about 25 aminoacids to about 130 amino acids, about 25 amino acids to about 125 aminoacids, about 25 amino acids to about 120 amino acids, about 25 aminoacids to about 115 amino acids, about 25 amino acids to about 110 aminoacids, about 25 amino acids to about 105 amino acids, about 25 aminoacids to about 100 amino acids, about 25 amino acids to about 95 aminoacids, about 25 amino acids to about 90 amino acids, about 25 aminoacids to about 85 amino acids, about 25 amino acids to about 80 aminoacids, about 25 amino acids to about 75 amino acids, about 25 aminoacids to about 70 amino acids, about 25 amino acids to about 65 aminoacids, about 25 amino acids to about 60 amino acids, about 25 aminoacids to about 55 amino acids, about 25 amino acids to about 50 aminoacids, about 25 amino acids to about 45 amino acids, about 25 aminoacids to about 40 amino acids, about 25 amino acids to about 35 aminoacids, about 25 amino acids to about 30 amino acids, about 30 aminoacids to about 200 amino acids, about 30 amino acids to about 195 aminoacids, about 30 amino acids to about 190 amino acids, about 30 aminoacids to about 185 amino acids, about 30 amino acids to about 180 aminoacids, about 30 amino acids to about 175 amino acids, about 30 aminoacids to about 170 amino acids, about 30 amino acids to about 165 aminoacids, about 30 amino acids to about 160 amino acids, about 30 aminoacids to about 155 amino acids, about 30 amino acids to about 150 aminoacids, about 30 amino acids to about 145 amino acids, about 30 aminoacids to about 140 amino acids, about 30 amino acids to about 135 aminoacids, about 30 amino acids to about 130 amino acids, about 30 aminoacids to about 125 amino acids, about 30 amino acids to about 120 aminoacids, about 30 amino acids to about 115 amino acids, about 30 aminoacids to about 110 amino acids, about 30 amino acids to about 105 aminoacids, about 30 amino acids to about 100 amino acids, about 30 aminoacids to about 95 amino acids, about 30 amino acids to about 90 aminoacids, about 30 amino acids to about 85 amino acids, about 30 aminoacids to about 80 amino acids, about 30 amino acids to about 75 aminoacids, about 30 amino acids to about 70 amino acids, about 30 aminoacids to about 65 amino acids, about 30 amino acids to about 60 aminoacids, about 30 amino acids to about 55 amino acids, about 30 aminoacids to about 50 amino acids, about 30 amino acids to about 45 aminoacids, about 30 amino acids to about 40 amino acids, about 30 aminoacids to about 35 amino acids, about 35 amino acids to about 200 aminoacids, about 35 amino acids to about 195 amino acids, about 35 aminoacids to about 190 amino acids, about 35 amino acids to about 185 aminoacids, about 35 amino acids to about 180 amino acids, about 35 aminoacids to about 175 amino acids, about 35 amino acids to about 170 aminoacids, about 35 amino acids to about 165 amino acids, about 35 aminoacids to about 160 amino acids, about 35 amino acids to about 155 aminoacids, about 35 amino acids to about 150 amino acids, about 35 aminoacids to about 145 amino acids, about 35 amino acids to about 140 aminoacids, about 35 amino acids to about 135 amino acids, about 35 aminoacids to about 130 amino acids, about 35 amino acids to about 125 aminoacids, about 35 amino acids to about 120 amino acids, about 35 aminoacids to about 115 amino acids, about 35 amino acids to about 110 aminoacids, about 35 amino acids to about 105 amino acids, about 35 aminoacids to about 100 amino acids, about 35 amino acids to about 95 aminoacids, about 35 amino acids to about 90 amino acids, about 35 aminoacids to about 85 amino acids, about 35 amino acids to about 80 aminoacids, about 35 amino acids to about 75 amino acids, about 35 aminoacids to about 70 amino acids, about 35 amino acids to about 65 aminoacids, about 35 amino acids to about 60 amino acids, about 35 aminoacids to about 55 amino acids, about 35 amino acids to about 50 aminoacids, about 35 amino acids to about 45 amino acids, about 35 aminoacids to about 40 amino acids, about 40 amino acids to about 200 aminoacids, about 40 amino acids to about 195 amino acids, about 40 aminoacids to about 190 amino acids, about 40 amino acids to about 185 aminoacids, about 40 amino acids to about 180 amino acids, about 40 aminoacids to about 175 amino acids, about 40 amino acids to about 170 aminoacids, about 40 amino acids to about 165 amino acids, about 40 aminoacids to about 160 amino acids, about 40 amino acids to about 155 aminoacids, about 40 amino acids to about 150 amino acids, about 40 aminoacids to about 145 amino acids, about 40 amino acids to about 140 aminoacids, about 40 amino acids to about 135 amino acids, about 40 aminoacids to about 130 amino acids, about 40 amino acids to about 125 aminoacids, about 40 amino acids to about 120 amino acids, about 40 aminoacids to about 115 amino acids, about 40 amino acids to about 110 aminoacids, about 40 amino acids to about 105 amino acids, about 40 aminoacids to about 100 amino acids, about 40 amino acids to about 95 aminoacids, about 40 amino acids to about 90 amino acids, about 40 aminoacids to about 85 amino acids, about 40 amino acids to about 80 aminoacids, about 40 amino acids to about 75 amino acids, about 40 aminoacids to about 70 amino acids, about 40 amino acids to about 65 aminoacids, about 40 amino acids to about 60 amino acids, about 40 aminoacids to about 55 amino acids, about 40 amino acids to about 50 aminoacids, about 40 amino acids to about 45 amino acids, about 45 aminoacids to about 200 amino acids, about 45 amino acids to about 195 aminoacids, about 45 amino acids to about 190 amino acids, about 45 aminoacids to about 185 amino acids, about 45 amino acids to about 180 aminoacids, about 45 amino acids to about 175 amino acids, about 45 aminoacids to about 170 amino acids, about 45 amino acids to about 165 aminoacids, about 45 amino acids to about 160 amino acids, about 45 aminoacids to about 155 amino acids, about 45 amino acids to about 150 aminoacids, about 45 amino acids to about 145 amino acids, about 45 aminoacids to about 140 amino acids, about 45 amino acids to about 135 aminoacids, about 45 amino acids to about 130 amino acids, about 45 aminoacids to about 125 amino acids, about 45 amino acids to about 120 aminoacids, about 45 amino acids to about 115 amino acids, about 45 aminoacids to about 110 amino acids, about 45 amino acids to about 105 aminoacids, about 45 amino acids to about 100 amino acids, about 45 aminoacids to about 95 amino acids, about 45 amino acids to about 90 aminoacids, about 45 amino acids to about 85 amino acids, about 45 aminoacids to about 80 amino acids, about 45 amino acids to about 75 aminoacids, about 45 amino acids to about 70 amino acids, about 45 aminoacids to about 65 amino acids, about 45 amino acids to about 60 aminoacids, about 45 amino acids to about 55 amino acids, about 45 aminoacids to about 50 amino acids, about 50 amino acids to about 200 aminoacids, about 50 amino acids to about 195 amino acids, about 50 aminoacids to about 190 amino acids, about 50 amino acids to about 185 aminoacids, about 50 amino acids to about 180 amino acids, about 50 aminoacids to about 175 amino acids, about 50 amino acids to about 170 aminoacids, about 50 amino acids to about 165 amino acids, about 50 aminoacids to about 160 amino acids, about 50 amino acids to about 155 aminoacids, about 50 amino acids to about 150 amino acids, about 50 aminoacids to about 145 amino acids, about 50 amino acids to about 140 aminoacids, about 50 amino acids to about 135 amino acids, about 50 aminoacids to about 130 amino acids, about 50 amino acids to about 125 aminoacids, about 50 amino acids to about 120 amino acids, about 50 aminoacids to about 115 amino acids, about 50 amino acids to about 110 aminoacids, about 50 amino acids to about 105 amino acids, about 50 aminoacids to about 100 amino acids, about 50 amino acids to about 95 aminoacids, about 50 amino acids to about 90 amino acids, about 50 aminoacids to about 85 amino acids, about 50 amino acids to about 80 aminoacids, about 50 amino acids to about 75 amino acids, about 50 aminoacids to about 70 amino acids, about 50 amino acids to about 65 aminoacids, about 50 amino acids to about 60 amino acids, about 50 aminoacids to about 55 amino acids, about 55 amino acids to about 200 aminoacids, about 55 amino acids to about 195 amino acids, about 55 aminoacids to about 190 amino acids, about 55 amino acids to about 185 aminoacids, about 55 amino acids to about 180 amino acids, about 55 aminoacids to about 175 amino acids, about 55 amino acids to about 170 aminoacids, about 55 amino acids to about 165 amino acids, about 55 aminoacids to about 160 amino acids, about 55 amino acids to about 155 aminoacids, about 55 amino acids to about 150 amino acids, about 55 aminoacids to about 145 amino acids, about 55 amino acids to about 140 aminoacids, about 55 amino acids to about 135 amino acids, about 55 aminoacids to about 130 amino acids, about 55 amino acids to about 125 aminoacids, about 55 amino acids to about 120 amino acids, about 55 aminoacids to about 115 amino acids, about 55 amino acids to about 110 aminoacids, about 55 amino acids to about 105 amino acids, about 55 aminoacids to about 100 amino acids, about 55 amino acids to about 95 aminoacids, about 55 amino acids to about 90 amino acids, about 55 aminoacids to about 85 amino acids, about 55 amino acids to about 80 aminoacids, about 55 amino acids to about 75 amino acids, about 55 aminoacids to about 70 amino acids, about 55 amino acids to about 65 aminoacids, about 55 amino acids to about 60 amino acids, about 60 aminoacids to about 200 amino acids, about 60 amino acids to about 195 aminoacids, about 60 amino acids to about 190 amino acids, about 60 aminoacids to about 185 amino acids, about 60 amino acids to about 180 aminoacids, about 60 amino acids to about 175 amino acids, about 60 aminoacids to about 170 amino acids, about 60 amino acids to about 165 aminoacids, about 60 amino acids to about 160 amino acids, about 60 aminoacids to about 155 amino acids, about 60 amino acids to about 150 aminoacids, about 60 amino acids to about 145 amino acids, about 60 aminoacids to about 140 amino acids, about 60 amino acids to about 135 aminoacids, about 60 amino acids to about 130 amino acids, about 60 aminoacids to about 125 amino acids, about 60 amino acids to about 120 aminoacids, about 60 amino acids to about 115 amino acids, about 60 aminoacids to about 110 amino acids, about 60 amino acids to about 105 aminoacids, about 60 amino acids to about 100 amino acids, about 60 aminoacids to about 95 amino acids, about 60 amino acids to about 90 aminoacids, about 60 amino acids to about 85 amino acids, about 60 aminoacids to about 80 amino acids, about 60 amino acids to about 75 aminoacids, about 60 amino acids to about 70 amino acids, about 60 aminoacids to about 65 amino acids, about 65 amino acids to about 200 aminoacids, about 65 amino acids to about 195 amino acids, about 65 aminoacids to about 190 amino acids, about 65 amino acids to about 185 aminoacids, about 65 amino acids to about 180 amino acids, about 65 aminoacids to about 175 amino acids, about 65 amino acids to about 170 aminoacids, about 65 amino acids to about 165 amino acids, about 65 aminoacids to about 160 amino acids, about 65 amino acids to about 155 aminoacids, about 65 amino acids to about 150 amino acids, about 65 aminoacids to about 145 amino acids, about 65 amino acids to about 140 aminoacids, about 65 amino acids to about 135 amino acids, about 65 aminoacids to about 130 amino acids, about 65 amino acids to about 125 aminoacids, about 65 amino acids to about 120 amino acids, about 65 aminoacids to about 115 amino acids, about 65 amino acids to about 110 aminoacids, about 65 amino acids to about 105 amino acids, about 65 aminoacids to about 100 amino acids, about 65 amino acids to about 95 aminoacids, about 65 amino acids to about 90 amino acids, about 65 aminoacids to about 85 amino acids, about 65 amino acids to about 80 aminoacids, about 65 amino acids to about 75 amino acids, about 65 aminoacids to about 70 amino acids, about 70 amino acids to about 200 aminoacids, about 70 amino acids to about 195 amino acids, about 70 aminoacids to about 190 amino acids, about 70 amino acids to about 185 aminoacids, about 70 amino acids to about 180 amino acids, about 70 aminoacids to about 175 amino acids, about 70 amino acids to about 170 aminoacids, about 70 amino acids to about 165 amino acids, about 70 aminoacids to about 160 amino acids, about 70 amino acids to about 155 aminoacids, about 70 amino acids to about 150 amino acids, about 70 aminoacids to about 145 amino acids, about 70 amino acids to about 140 aminoacids, about 70 amino acids to about 135 amino acids, about 70 aminoacids to about 130 amino acids, about 70 amino acids to about 125 aminoacids, about 70 amino acids to about 120 amino acids, about 70 aminoacids to about 115 amino acids, about 70 amino acids to about 110 aminoacids, about 70 amino acids to about 105 amino acids, about 70 aminoacids to about 100 amino acids, about 70 amino acids to about 95 aminoacids, about 70 amino acids to about 90 amino acids, about 70 aminoacids to about 85 amino acids, about 70 amino acids to about 80 aminoacids, about 70 amino acids to about 75 amino acids, about 75 aminoacids to about 200 amino acids, about 75 amino acids to about 195 aminoacids, about 75 amino acids to about 190 amino acids, about 75 aminoacids to about 185 amino acids, about 75 amino acids to about 180 aminoacids, about 75 amino acids to about 175 amino acids, about 75 aminoacids to about 170 amino acids, about 75 amino acids to about 165 aminoacids, about 75 amino acids to about 160 amino acids, about 75 aminoacids to about 155 amino acids, about 75 amino acids to about 150 aminoacids, about 75 amino acids to about 145 amino acids, about 75 aminoacids to about 140 amino acids, about 75 amino acids to about 135 aminoacids, about 75 amino acids to about 130 amino acids, about 75 aminoacids to about 125 amino acids, about 75 amino acids to about 120 aminoacids, about 75 amino acids to about 115 amino acids, about 75 aminoacids to about 110 amino acids, about 75 amino acids to about 105 aminoacids, about 75 amino acids to about 100 amino acids, about 75 aminoacids to about 95 amino acids, about 75 amino acids to about 90 aminoacids, about 75 amino acids to about 85 amino acids, about 75 aminoacids to about 80 amino acids, about 80 amino acids to about 200 aminoacids, about 80 amino acids to about 195 amino acids, about 80 aminoacids to about 190 amino acids, about 80 amino acids to about 185 aminoacids, about 80 amino acids to about 180 amino acids, about 80 aminoacids to about 175 amino acids, about 80 amino acids to about 170 aminoacids, about 80 amino acids to about 165 amino acids, about 80 aminoacids to about 160 amino acids, about 80 amino acids to about 155 aminoacids, about 80 amino acids to about 150 amino acids, about 80 aminoacids to about 145 amino acids, about 80 amino acids to about 140 aminoacids, about 80 amino acids to about 135 amino acids, about 80 aminoacids to about 130 amino acids, about 80 amino acids to about 125 aminoacids, about 80 amino acids to about 120 amino acids, about 80 aminoacids to about 115 amino acids, about 80 amino acids to about 110 aminoacids, about 80 amino acids to about 105 amino acids, about 80 aminoacids to about 100 amino acids, about 80 amino acids to about 95 aminoacids, about 80 amino acids to about 90 amino acids, about 80 aminoacids to about 85 amino acids, about 85 amino acids to about 200 aminoacids, about 85 amino acids to about 195 amino acids, about 85 aminoacids to about 190 amino acids, about 85 amino acids to about 185 aminoacids, about 85 amino acids to about 180 amino acids, about 85 aminoacids to about 175 amino acids, about 85 amino acids to about 170 aminoacids, about 85 amino acids to about 165 amino acids, about 85 aminoacids to about 160 amino acids, about 85 amino acids to about 155 aminoacids, about 85 amino acids to about 150 amino acids, about 85 aminoacids to about 145 amino acids, about 85 amino acids to about 140 aminoacids, about 85 amino acids to about 135 amino acids, about 85 aminoacids to about 130 amino acids, about 85 amino acids to about 125 aminoacids, about 85 amino acids to about 120 amino acids, about 85 aminoacids to about 115 amino acids, about 85 amino acids to about 110 aminoacids, about 85 amino acids to about 105 amino acids, about 85 aminoacids to about 100 amino acids, about 85 amino acids to about 95 aminoacids, about 85 amino acids to about 90 amino acids, about 90 aminoacids to about 200 amino acids, about 90 amino acids to about 195 aminoacids, about 90 amino acids to about 190 amino acids, about 90 aminoacids to about 185 amino acids, about 90 amino acids to about 180 aminoacids, about 90 amino acids to about 175 amino acids, about 90 aminoacids to about 170 amino acids, about 90 amino acids to about 165 aminoacids, about 90 amino acids to about 160 amino acids, about 90 aminoacids to about 155 amino acids, about 90 amino acids to about 150 aminoacids, about 90 amino acids to about 145 amino acids, about 90 aminoacids to about 140 amino acids, about 90 amino acids to about 135 aminoacids, about 90 amino acids to about 130 amino acids, about 90 aminoacids to about 125 amino acids, about 90 amino acids to about 120 aminoacids, about 90 amino acids to about 115 amino acids, about 90 aminoacids to about 110 amino acids, about 90 amino acids to about 105 aminoacids, about 90 amino acids to about 100 amino acids, about 90 aminoacids to about 95 amino acids, about 95 amino acids to about 200 aminoacids, about 95 amino acids to about 195 amino acids, about 95 aminoacids to about 190 amino acids, about 95 amino acids to about 185 aminoacids, about 95 amino acids to about 180 amino acids, about 95 aminoacids to about 175 amino acids, about 95 amino acids to about 170 aminoacids, about 95 amino acids to about 165 amino acids, about 95 aminoacids to about 160 amino acids, about 95 amino acids to about 155 aminoacids, about 95 amino acids to about 150 amino acids, about 95 aminoacids to about 145 amino acids, about 95 amino acids to about 140 aminoacids, about 95 amino acids to about 135 amino acids, about 95 aminoacids to about 130 amino acids, about 95 amino acids to about 125 aminoacids, about 95 amino acids to about 120 amino acids, about 95 aminoacids to about 115 amino acids, about 95 amino acids to about 110 aminoacids, about 95 amino acids to about 105 amino acids, about 95 aminoacids to about 100 amino acids, about 100 amino acids to about 200 aminoacids, about 100 amino acids to about 195 amino acids, about 100 aminoacids to about 190 amino acids, about 100 amino acids to about 185 aminoacids, about 100 amino acids to about 180 amino acids, about 100 aminoacids to about 175 amino acids, about 100 amino acids to about 170 aminoacids, about 100 amino acids to about 165 amino acids, about 100 aminoacids to about 160 amino acids, about 100 amino acids to about 155 aminoacids, about 100 amino acids to about 150 amino acids, about 100 aminoacids to about 145 amino acids, about 100 amino acids to about 140 aminoacids, about 100 amino acids to about 135 amino acids, about 100 aminoacids to about 130 amino acids, about 100 amino acids to about 125 aminoacids, about 100 amino acids to about 120 amino acids, about 100 aminoacids to about 115 amino acids, about 100 amino acids to about 110 aminoacids, about 100 amino acids to about 105 amino acids, about 105 aminoacids to about 200 amino acids, about 105 amino acids to about 195 aminoacids, about 105 amino acids to about 190 amino acids, about 105 aminoacids to about 185 amino acids, about 105 amino acids to about 180 aminoacids, about 105 amino acids to about 175 amino acids, about 105 aminoacids to about 170 amino acids, about 105 amino acids to about 165 aminoacids, about 105 amino acids to about 160 amino acids, about 105 aminoacids to about 155 amino acids, about 105 amino acids to about 150 aminoacids, about 105 amino acids to about 145 amino acids, about 105 aminoacids to about 140 amino acids, about 105 amino acids to about 135 aminoacids, about 105 amino acids to about 130 amino acids, about 105 aminoacids to about 125 amino acids, about 105 amino acids to about 120 aminoacids, about 105 amino acids to about 115 amino acids, about 105 aminoacids to about 110 amino acids, about 110 amino acids to about 200 aminoacids, about 110 amino acids to about 195 amino acids, about 110 aminoacids to about 190 amino acids, about 110 amino acids to about 185 aminoacids, about 110 amino acids to about 180 amino acids, about 110 aminoacids to about 175 amino acids, about 110 amino acids to about 170 aminoacids, about 110 amino acids to about 165 amino acids, about 110 aminoacids to about 160 amino acids, about 110 amino acids to about 155 aminoacids, about 110 amino acids to about 150 amino acids, about 110 aminoacids to about 145 amino acids, about 110 amino acids to about 140 aminoacids, about 110 amino acids to about 135 amino acids, about 110 aminoacids to about 130 amino acids, about 110 amino acids to about 125 aminoacids, about 110 amino acids to about 120 amino acids, about 110 aminoacids to about 115 amino acids, about 115 amino acids to about 200 aminoacids, about 115 amino acids to about 195 amino acids, about 115 aminoacids to about 190 amino acids, about 115 amino acids to about 185 aminoacids, about 115 amino acids to about 180 amino acids, about 115 aminoacids to about 175 amino acids, about 115 amino acids to about 170 aminoacids, about 115 amino acids to about 165 amino acids, about 115 aminoacids to about 160 amino acids, about 115 amino acids to about 155 aminoacids, about 115 amino acids to about 150 amino acids, about 115 aminoacids to about 145 amino acids, about 115 amino acids to about 140 aminoacids, about 115 amino acids to about 135 amino acids, about 115 aminoacids to about 130 amino acids, about 115 amino acids to about 125 aminoacids, about 115 amino acids to about 120 amino acids, about 120 aminoacids to about 200 amino acids, about 120 amino acids to about 195 aminoacids, about 120 amino acids to about 190 amino acids, about 120 aminoacids to about 185 amino acids, about 120 amino acids to about 180 aminoacids, about 120 amino acids to about 175 amino acids, about 120 aminoacids to about 170 amino acids, about 120 amino acids to about 165 aminoacids, about 120 amino acids to about 160 amino acids, about 120 aminoacids to about 155 amino acids, about 120 amino acids to about 150 aminoacids, about 120 amino acids to about 145 amino acids, about 120 aminoacids to about 140 amino acids, about 120 amino acids to about 135 aminoacids, about 120 amino acids to about 130 amino acids, about 120 aminoacids to about 125 amino acids, about 125 amino acids to about 200 aminoacids, about 125 amino acids to about 195 amino acids, about 125 aminoacids to about 190 amino acids, about 125 amino acids to about 185 aminoacids, about 125 amino acids to about 180 amino acids, about 125 aminoacids to about 175 amino acids, about 125 amino acids to about 170 aminoacids, about 125 amino acids to about 165 amino acids, about 125 aminoacids to about 160 amino acids, about 125 amino acids to about 155 aminoacids, about 125 amino acids to about 150 amino acids, about 125 aminoacids to about 145 amino acids, about 125 amino acids to about 140 aminoacids, about 125 amino acids to about 135 amino acids, about 125 aminoacids to about 130 amino acids, about 130 amino acids to about 200 aminoacids, about 130 amino acids to about 195 amino acids, about 130 aminoacids to about 190 amino acids, about 130 amino acids to about 185 aminoacids, about 130 amino acids to about 180 amino acids, about 130 aminoacids to about 175 amino acids, about 130 amino acids to about 170 aminoacids, about 130 amino acids to about 165 amino acids, about 130 aminoacids to about 160 amino acids, about 130 amino acids to about 155 aminoacids, about 130 amino acids to about 150 amino acids, about 130 aminoacids to about 145 amino acids, about 130 amino acids to about 140 aminoacids, about 130 amino acids to about 135 amino acids, about 135 aminoacids to about 200 amino acids, about 135 amino acids to about 195 aminoacids, about 135 amino acids to about 190 amino acids, about 135 aminoacids to about 185 amino acids, about 135 amino acids to about 180 aminoacids, about 135 amino acids to about 175 amino acids, about 135 aminoacids to about 170 amino acids, about 135 amino acids to about 165 aminoacids, about 135 amino acids to about 160 amino acids, about 135 aminoacids to about 155 amino acids, about 135 amino acids to about 150 aminoacids, about 135 amino acids to about 145 amino acids, about 135 aminoacids to about 140 amino acids, about 140 amino acids to about 200 aminoacids, about 140 amino acids to about 195 amino acids, about 140 aminoacids to about 190 amino acids, about 140 amino acids to about 185 aminoacids, about 140 amino acids to about 180 amino acids, about 140 aminoacids to about 175 amino acids, about 140 amino acids to about 170 aminoacids, about 140 amino acids to about 165 amino acids, about 140 aminoacids to about 160 amino acids, about 140 amino acids to about 155 aminoacids, about 140 amino acids to about 150 amino acids, about 40 aminoacids to about 145 amino acids, about 145 amino acids to about 200 aminoacids, about 145 amino acids to about 195 amino acids, about 145 aminoacids to about 190 amino acids, about 145 amino acids to about 185 aminoacids, about 145 amino acids to about 180 amino acids, about 145 aminoacids to about 175 amino acids, about 145 amino acids to about 170 aminoacids, about 145 amino acids to about 165 amino acids, about 145 aminoacids to about 160 amino acids, about 145 amino acids to about 155 aminoacids, about 145 amino acids to about 150 amino acids, about 150 aminoacids to about 200 amino acids, about 150 amino acids to about 195 aminoacids, about 150 amino acids to about 190 amino acids, about 150 aminoacids to about 185 amino acids, about 150 amino acids to about 180 aminoacids, about 150 amino acids to about 175 amino acids, about 150 aminoacids to about 170 amino acids, about 150 amino acids to about 165 aminoacids, about 150 amino acids to about 160 amino acids, about 150 aminoacids to about 155 amino acids, about 155 amino acids to about 200 aminoacids, about 155 amino acids to about 195 amino acids, about 155 aminoacids to about 190 amino acids, about 155 amino acids to about 185 aminoacids, about 155 amino acids to about 180 amino acids, about 155 aminoacids to about 175 amino acids, about 155 amino acids to about 170 aminoacids, about 155 amino acids to about 165 amino acids, about 155 aminoacids to about 160 amino acids, about 160 amino acids to about 200 aminoacids, about 160 amino acids to about 195 amino acids, about 160 aminoacids to about 190 amino acids, about 160 amino acids to about 185 aminoacids, about 160 amino acids to about 180 amino acids, about 160 aminoacids to about 175 amino acids, about 160 amino acids to about 170 aminoacids, about 160 amino acids to about 165 amino acids, about 165 aminoacids to about 200 amino acids, about 165 amino acids to about 195 aminoacids, about 165 amino acids to about 190 amino acids, about 165 aminoacids to about 185 amino acids, about 165 amino acids to about 180 aminoacids, about 165 amino acids to about 175 amino acids, about 165 aminoacids to about 170 amino acids, about 170 amino acids to about 200 aminoacids, about 170 amino acids to about 195 amino acids, about 170 aminoacids to about 190 amino acids, about 170 amino acids to about 185 aminoacids, about 170 amino acids to about 180 amino acids, about 170 aminoacids to about 175 amino acids, about 175 amino acids to about 200 aminoacids, about 175 amino acids to about 195 amino acids, about 175 aminoacids to about 190 amino acids, about 175 amino acids to about 185 aminoacids, about 175 amino acids to about 180 amino acids, about 180 aminoacids to about 200 amino acids, about 180 amino acids to about 195 aminoacids, about 180 amino acids to about 190 amino acids, about 180 aminoacids to about 185 amino acids, about 185 amino acids to about 200 aminoacids, about 185 amino acids to about 195 amino acids, about 185 aminoacids to about 190 amino acids, about 190 amino acids to about 200 aminoacids, about 190 amino acids to about 195 amino acids, about 195 aminoacids to about 200 amino acids, deleted. In some embodiments where twoor more amino acids are deleted from the sequence of a wildtype,full-length otoferlin protein, the two or more deleted amino acids canbe contiguous in the sequence of the wildtype, full-length protein. Inother examples where two or more amino acids are deleted from thesequence of a wildtype, full-length otoferlin protein, the two or moredeleted amino acids are not contiguous in the sequence of the wildtype,full-length protein. One skilled in the art would appreciate that aminoacids that are not conserved between wildtype, full-length otoferlinproteins from different species can be deleted without losing activity,while those amino acids that are conserved between wildtype, full-lengthotoferlin proteins from different species should not be deleted as theyare more likely (than amino acids that are not conserved betweendifferent species) to be involved in activity.

In some examples, an active otoferlin protein can, e.g., include asequence of a wildtype, full-length otoferlin protein that has between 1amino acid to about 100 amino acids, 1 amino acid to about 95 aminoacids, 1 amino acid to about 90 amino acids, 1 amino acid to about 85amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about75 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid toabout 65 amino acids, 1 amino acid to about 60 amino acids, 1 amino acidto about 55 amino acids, 1 amino acid to about 50 amino acids, 1 aminoacid to about 45 amino acids, 1 amino acid to about 40 amino acids, 1amino acid to about 35 amino acids, 1 amino acid to about 30 aminoacids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20amino acids, 1 amino acid to about 15 amino acids, 1 amino acid to about10 amino acids, 1 amino acid to about 9 amino acids, 1 amino acid toabout 8 amino acids, 1 amino acid to about 7 amino acids, 1 amino acidto about 6 amino acids, 1 amino acid to about 5 amino acids, 1 aminoacid to about 4 amino acids, 1 amino acid to about 3 amino acids, about2 amino acids to about 100 amino acids, about 2 amino acid to about 95amino acids, about 2 amino acids to about 90 amino acids, about 2 aminoacids to about 85 amino acids, about 2 amino acids to about 80 aminoacids, about 2 amino acids to about 75 amino acids, about 2 amino acidsto about 70 amino acids, about 2 amino acids to about 65 amino acids,about 2 amino acids to about 60 amino acids, about 2 amino acids toabout 55 amino acids, about 2 amino acids to about 50 amino acids, about2 amino acids to about 45 amino acids, about 2 amino acids to about 40amino acids, about 2 amino acids to about 35 amino acids, about 2 aminoacids to about 30 amino acids, about 2 amino acids to about 25 aminoacids, about 2 amino acids to about 20 amino acids, about 2 amino acidsto about 15 amino acids, about 2 amino acids to about 10 amino acids,about 2 amino acids to about 9 amino acids, about 2 amino acids to about8 amino acids, about 2 amino acids to about 7 amino acids, about 2 aminoacids to about 6 amino acids, about 2 amino acids to about 5 aminoacids, about 2 amino acids to about 4 amino acids, about 3 amino acidsto about 100 amino acids, about 3 amino acid to about 95 amino acids,about 3 amino acids to about 90 amino acids, about 3 amino acids toabout 85 amino acids, about 3 amino acids to about 80 amino acids, about3 amino acids to about 75 amino acids, about 3 amino acids to about 70amino acids, about 3 amino acids to about 65 amino acids, about 3 aminoacids to about 60 amino acids, about 3 amino acids to about 55 aminoacids, about 3 amino acids to about 50 amino acids, about 3 amino acidsto about 45 amino acids, about 3 amino acids to about 40 amino acids,about 3 amino acids to about 35 amino acids, about 3 amino acids toabout 30 amino acids, about 3 amino acids to about 25 amino acids, about3 amino acids to about 20 amino acids, about 3 amino acids to about 15amino acids, about 3 amino acids to about 10 amino acids, about 3 aminoacids to about 9 amino acids, about 3 amino acids to about 8 aminoacids, about 3 amino acids to about 7 amino acids, about 3 amino acidsto about 6 amino acids, about 3 amino acids to about 5 amino acids,about 4 amino acids to about 100 amino acids, about 4 amino acid toabout 95 amino acids, about 4 amino acids to about 90 amino acids, about4 amino acids to about 85 amino acids, about 4 amino acids to about 80amino acids, about 4 amino acids to about 75 amino acids, about 4 aminoacids to about 70 amino acids, about 4 amino acids to about 65 aminoacids, about 4 amino acids to about 60 amino acids, about 4 amino acidsto about 55 amino acids, about 4 amino acids to about 50 amino acids,about 4 amino acids to about 45 amino acids, about 4 amino acids toabout 40 amino acids, about 4 amino acids to about 35 amino acids, about4 amino acids to about 30 amino acids, about 4 amino acids to about 25amino acids, about 4 amino acids to about 20 amino acids, about 4 aminoacids to about 15 amino acids, about 4 amino acids to about 10 aminoacids, about 4 amino acids to about 9 amino acids, about 4 amino acidsto about 8 amino acids, about 4 amino acids to about 7 amino acids,about 4 amino acids to about 6 amino acids, about 5 amino acids to about100 amino acids, about 5 amino acid to about 95 amino acids, about 5amino acids to about 90 amino acids, about 5 amino acids to about 85amino acids, about 5 amino acids to about 80 amino acids, about 5 aminoacids to about 75 amino acids, about 5 amino acids to about 70 aminoacids, about 5 amino acids to about 65 amino acids, about 5 amino acidsto about 60 amino acids, about 5 amino acids to about 55 amino acids,about 5 amino acids to about 50 amino acids, about 5 amino acids toabout 45 amino acids, about 5 amino acids to about 40 amino acids, about5 amino acids to about 35 amino acids, about 5 amino acids to about 30amino acids, about 5 amino acids to about 25 amino acids, about 5 aminoacids to about 20 amino acids, about 5 amino acids to about 15 aminoacids, about 5 amino acids to about 10 amino acids, about 5 amino acidsto about 9 amino acids, about 5 amino acids to about 8 amino acids,about 5 amino acids to about 7 amino acids, about 6 amino acids to about100 amino acids, about 6 amino acid to about 95 amino acids, about 6amino acids to about 90 amino acids, about 6 amino acids to about 85amino acids, about 6 amino acids to about 80 amino acids, about 6 aminoacids to about 75 amino acids, about 6 amino acids to about 70 aminoacids, about 6 amino acids to about 65 amino acids, about 6 amino acidsto about 60 amino acids, about 6 amino acids to about 55 amino acids,about 6 amino acids to about 50 amino acids, about 6 amino acids toabout 45 amino acids, about 6 amino acids to about 40 amino acids, about6 amino acids to about 35 amino acids, about 6 amino acids to about 30amino acids, about 6 amino acids to about 25 amino acids, about 6 aminoacids to about 20 amino acids, about 6 amino acids to about 15 aminoacids, about 6 amino acids to about 10 amino acids, about 6 amino acidsto about 9 amino acids, about 6 amino acids to about 8 amino acids,about 7 amino acids to about 100 amino acids, about 7 amino acid toabout 95 amino acids, about 7 amino acids to about 90 amino acids, about7 amino acids to about 85 amino acids, about 7 amino acids to about 80amino acids, about 7 amino acids to about 75 amino acids, about 7 aminoacids to about 70 amino acids, about 7 amino acids to about 65 aminoacids, about 7 amino acids to about 60 amino acids, about 7 amino acidsto about 55 amino acids, about 7 amino acids to about 50 amino acids,about 7 amino acids to about 45 amino acids, about 7 amino acids toabout 40 amino acids, about 7 amino acids to about 35 amino acids, about7 amino acids to about 30 amino acids, about 7 amino acids to about 25amino acids, about 7 amino acids to about 20 amino acids, about 7 aminoacids to about 15 amino acids, about 7 amino acids to about 10 aminoacids, about 7 amino acids to about 9 amino acids, about 8 amino acidsto about 100 amino acids, about 8 amino acid to about 95 amino acids,about 8 amino acids to about 90 amino acids, about 8 amino acids toabout 85 amino acids, about 8 amino acids to about 80 amino acids, about8 amino acids to about 75 amino acids, about 8 amino acids to about 70amino acids, about 8 amino acids to about 65 amino acids, about 8 aminoacids to about 60 amino acids, about 8 amino acids to about 55 aminoacids, about 8 amino acids to about 50 amino acids, about 8 amino acidsto about 45 amino acids, about 8 amino acids to about 40 amino acids,about 8 amino acids to about 35 amino acids, about 8 amino acids toabout 30 amino acids, about 8 amino acids to about 25 amino acids, about8 amino acids to about 20 amino acids, about 8 amino acids to about 15amino acids, about 8 amino acids to about 10 amino acids, about 10 aminoacids to about 100 amino acids, about 10 amino acid to about 95 aminoacids, about 10 amino acids to about 90 amino acids, about 10 aminoacids to about 85 amino acids, about 10 amino acids to about 80 aminoacids, about 10 amino acids to about 75 amino acids, about 10 aminoacids to about 70 amino acids, about 10 amino acids to about 65 aminoacids, about 10 amino acids to about 60 amino acids, about 10 aminoacids to about 55 amino acids, about 10 amino acids to about 50 aminoacids, about 10 amino acids to about 45 amino acids, about 10 aminoacids to about 40 amino acids, about 10 amino acids to about 35 aminoacids, about 10 amino acids to about 30 amino acids, about 10 aminoacids to about 25 amino acids, about 10 amino acids to about 20 aminoacids, about 10 amino acids to about 15 amino acids, about 20 aminoacids to about 100 amino acids, about 20 amino acid to about 95 aminoacids, about 20 amino acids to about 90 amino acids, about 20 aminoacids to about 85 amino acids, about 20 amino acids to about 80 aminoacids, about 20 amino acids to about 75 amino acids, about 20 aminoacids to about 70 amino acids, about 20 amino acids to about 65 aminoacids, about 20 amino acids to about 60 amino acids, about 20 aminoacids to about 55 amino acids, about 20 amino acids to about 50 aminoacids, about 20 amino acids to about 45 amino acids, about 20 aminoacids to about 40 amino acids, about 20 amino acids to about 35 aminoacids, about 20 amino acids to about 30 amino acids, about 20 aminoacids to about 25 amino acids, about 30 amino acids to about 100 aminoacids, about 30 amino acid to about 95 amino acids, about 30 amino acidsto about 90 amino acids, about 30 amino acids to about 85 amino acids,about 30 amino acids to about 80 amino acids, about 30 amino acids toabout 75 amino acids, about 30 amino acids to about 70 amino acids,about 30 amino acids to about 65 amino acids, about 30 amino acids toabout 60 amino acids, about 30 amino acids to about 55 amino acids,about 30 amino acids to about 50 amino acids, about 30 amino acids toabout 45 amino acids, about 30 amino acids to about 40 amino acids,about 30 amino acids to about 35 amino acids, about 40 amino acids toabout 100 amino acids, about 40 amino acid to about 95 amino acids,about 40 amino acids to about 90 amino acids, about 40 amino acids toabout 85 amino acids, about 40 amino acids to about 80 amino acids,about 40 amino acids to about 75 amino acids, about 40 amino acids toabout 70 amino acids, about 40 amino acids to about 65 amino acids,about 40 amino acids to about 60 amino acids, about 40 amino acids toabout 55 amino acids, about 40 amino acids to about 50 amino acids,about 40 amino acids to about 45 amino acids, about 50 amino acids toabout 100 amino acids, about 50 amino acid to about 95 amino acids,about 50 amino acids to about 90 amino acids, about 50 amino acids toabout 85 amino acids, about 50 amino acids to about 80 amino acids,about 50 amino acids to about 75 amino acids, about 50 amino acids toabout 70 amino acids, about 50 amino acids to about 65 amino acids,about 50 amino acids to about 60 amino acids, about 50 amino acids toabout 55 amino acids, about 60 amino acids to about 100 amino acids,about 60 amino acid to about 95 amino acids, about 60 amino acids toabout 90 amino acids, about 60 amino acids to about 85 amino acids,about 60 amino acids to about 80 amino acids, about 60 amino acids toabout 75 amino acids, about 60 amino acids to about 70 amino acids,about 60 amino acids to about 65 amino acids, about 70 amino acids toabout 100 amino acids, about 70 amino acid to about 95 amino acids,about 70 amino acids to about 90 amino acids, about 70 amino acids toabout 85 amino acids, about 70 amino acids to about 80 amino acids,about 70 amino acids to about 75 amino acids, about 80 amino acids toabout 100 amino acids, about 80 amino acid to about 95 amino acids,about 80 amino acids to about 90 amino acids, about 80 amino acids toabout 85 amino acids, about 90 amino acids to about 100 amino acids,about 90 amino acids to about 95 amino acids, or about 95 amino acids toabout 100 amino acids, removed from its N-terminus and/or from itsC-terminus.

In some embodiments, an active otoferlin protein can, e.g., include thesequence of a wildtype, full-length otoferlin protein where 1 amino acidto 50 amino acids, 1 amino acid to 45 amino acids, 1 amino acid to 40amino acids, 1 amino acid to 35 amino acids, 1 amino acid to 30 aminoacids, 1 amino acid to 25 amino acids, 1 amino acid to 20 amino acids, 1amino acid to 15 amino acids, 1 amino acid to 10 amino acids, 1 aminoacid to 9 amino acids, 1 amino acid to 8 amino acids, 1 amino acid to 7amino acids, 1 amino acid to 6 amino acids, 1 amino acid to 5 aminoacids, 1 amino acid to 4 amino acids, 1 amino acid to 3 amino acids,about 2 amino acids to 50 amino acids, about 2 amino acids to 45 aminoacids, about 2 amino acids to 40 amino acids, about 2 amino acids to 35amino acids, about 2 amino acids to 30 amino acids, about 2 amino acidsto 25 amino acids, about 2 amino acids to 20 amino acids, about 2 aminoacids to 15 amino acids, about 2 amino acids to 10 amino acids, about 2amino acids to 9 amino acids, about 2 amino acids to 8 amino acids,about 2 amino acids to 7 amino acids, about 2 amino acids to 6 aminoacids, about 2 amino acids to 5 amino acids, about 2 amino acids to 4amino acids, about 3 amino acids to 50 amino acids, about 3 amino acidsto 45 amino acids, about 3 amino acids to 40 amino acids, about 3 aminoacids to 35 amino acids, about 3 amino acids to 30 amino acids, about 3amino acids to 25 amino acids, about 3 amino acids to 20 amino acids,about 3 amino acids to 15 amino acids, about 3 amino acids to 10 aminoacids, about 3 amino acids to 9 amino acids, about 3 amino acids to 8amino acids, about 3 amino acids to 7 amino acids, about 3 amino acidsto 6 amino acids, about 3 amino acids to 5 amino acids, about 4 aminoacids to 50 amino acids, about 4 amino acids to 45 amino acids, about 4amino acids to 40 amino acids, about 4 amino acids to 35 amino acids,about 4 amino acids to 30 amino acids, about 4 amino acids to 25 aminoacids, about 4 amino acids to 20 amino acids, about 4 amino acids to 15amino acids, about 4 amino acids to 10 amino acids, about 4 amino acidsto 9 amino acids, about 4 amino acids to 8 amino acids, about 4 aminoacids to 7 amino acids, about 4 amino acids to 6 amino acids, about 5amino acids to 50 amino acids, about 5 amino acids to 45 amino acids,about 5 amino acids to 40 amino acids, about 5 amino acids to 35 aminoacids, about 5 amino acids to 30 amino acids, about 5 amino acids to 25amino acids, about 5 amino acids to 20 amino acids, about 5 amino acidsto 15 amino acids, about 5 amino acids to 10 amino acids, about 5 aminoacids to 9 amino acids, about 5 amino acids to 8 amino acids, about 5amino acids to 7 amino acids, about 6 amino acids to 50 amino acids,about 6 amino acids to 45 amino acids, about 6 amino acids to 40 aminoacids, about 6 amino acids to 35 amino acids, about 6 amino acids to 30amino acids, about 6 amino acids to 25 amino acids, about 6 amino acidsto 20 amino acids, about 6 amino acids to 15 amino acids, about 6 aminoacids to 10 amino acids, about 6 amino acids to 9 amino acids, about 6amino acids to 8 amino acids, about 7 amino acids to 50 amino acids,about 7 amino acids to 45 amino acids, about 7 amino acids to 40 aminoacids, about 7 amino acids to 35 amino acids, about 7 amino acids to 30amino acids, about 7 amino acids to 25 amino acids, about 7 amino acidsto 20 amino acids, about 7 amino acids to 15 amino acids, about 7 aminoacids to 10 amino acids, about 7 amino acids to 9 amino acids, about 8amino acids to 50 amino acids, about 8 amino acids to 45 amino acids,about 8 amino acids to 40 amino acids, about 8 amino acids to 35 aminoacids, about 8 amino acids to 30 amino acids, about 8 amino acids to 25amino acids, about 8 amino acids to 20 amino acids, about 8 amino acidsto 15 amino acids, about 8 amino acids to 10 amino acids, about 10 aminoacids to 50 amino acids, about 10 amino acids to 45 amino acids, about10 amino acids to 40 amino acids, about 10 amino acids to 35 aminoacids, about 10 amino acids to 30 amino acids, about 10 amino acids to25 amino acids, about 10 amino acids to 20 amino acids, about 10 aminoacids to 15 amino acids, about 15 amino acids to 50 amino acids, about15 amino acids to 45 amino acids, about 15 amino acids to 40 aminoacids, about 15 amino acids to 35 amino acids, about 15 amino acids to30 amino acids, about 15 amino acids to 25 amino acids, about 15 aminoacids to 20 amino acids, about 20 amino acids to 50 amino acids, about20 amino acids to 45 amino acids, about 20 amino acids to 40 aminoacids, about 20 amino acids to 35 amino acids, about 20 amino acids to30 amino acids, about 20 amino acids to 25 amino acids, about 25 aminoacids to 50 amino acids, about 25 amino acids to 45 amino acids, about25 amino acids to 40 amino acids, about 25 amino acids to 35 aminoacids, about 25 amino acids to 30 amino acids, about 30 amino acids to50 amino acids, about 30 amino acids to 45 amino acids, about 30 aminoacids to 40 amino acids, about 30 amino acids to 35 amino acids, about35 amino acids to 50 amino acids, about 35 amino acids to 45 aminoacids, about 35 amino acids to 40 amino acids, about 40 amino acids to50 amino acids, about 40 amino acids to 45 amino acids, or about 45amino acids to about 50 amino acids, are inserted. In some examples, theinserted amino acid(s) can be inserted as a contiguous sequence into thesequence of a wildtype, full-length protein. In some examples, the aminoacid(s) are not inserted as a contiguous sequence into the sequence of awildtype, full-length protein. As can be appreciated in the art, theamino acid(s) can be inserted into a portion of the sequence of awildtype, full-length protein that is not well-conserved betweenspecies.

Ranges: throughout this disclosure, various aspects of the disclosurecan be presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of thedisclosure. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Asanother example, a range such as 95-99% identity includes something with95%, 96%, 97%, 98% or 99% identity, and includes subranges such as96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This appliesregardless of the breadth of the range.

In one aspect the present disclosure provides a recombinant AAV vectorof SEQ ID NO: 96. In one aspect the present disclosure provides arecombinant AAV vector of SEQ ID NO: 105.

In one aspect the present disclosure provides a recombinant AAV vectorthat comprises, in order of 5′ to 3′: a 5′ ITR sequence of SEQ ID NO:97; a CAG promoter comprising a CMV early enhancer element of SEQ ID NO:98, a chicken beta actin gene sequence of SEQ ID NO: 99, and a chimericintron of SEQ ID NO: 100; a 5′OTOF coding region that comprises exons 1to (and through) 21 of OTOF cDNA; a SD intron sequence of SEQ ID NO:102; an AK recombinogenic sequence of SEQ ID NO: 103; and a 3′ ITRsequence of SEQ ID NO: 104. In some embodiments, the 5′OTOF codingregion is SEQ ID NO: 101. In some embodiments, the 5′OTOF coding regionis at least 70%, at least 80%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, at least 99.9% or 100%identical to SEQ ID NO: 101, and encodes the same amino acid sequence asencoded by SEQ ID NO: 101.

In one aspect the present disclosure provides a recombinant AAV vectorthat comprises, in order of 5′ to 3′: a 5′ ITR sequence of SEQ ID NO:97; an AK recombinogenic sequence of SEQ ID NO: 103; a SA intronsequence of SEQ ID NO: 106; a 3′ OTOF coding region that comprises exons22 to (and through) exon 48 of OTOF cDNA; a bgH polyA sequence of SEQ IDNO: 108; and a 3′ ITR sequence of SEQ ID NO: 104. In some embodiments,the 3′OTOF coding region is SEQ ID NO: 107. In some embodiments, the3′OTOF coding region is at least 70%, at least 80%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, atleast 99.9% or 100% identical to SEQ ID NO: 107, and encodes the sameamino acid sequence as encoded by SEQ ID NO: 107.

In one aspect the present disclosure provides an rAAV particlecomprising one of the aforementioned recombinant AAV vectorsencapsidated by an Anc80 capsid. In some embodiment, the Anc80 capsidcomprises a polypeptide of SEQ ID NO: 109. In one aspect the presentdisclosure provides a composition comprising a first rAAV particlecomprising a recombinant AAV vector of SEQ ID NO: 96 and a second rAAVparticle comprising a recombinant AAV vector of SEQ ID NO: 105. In someembodiments the recombinant AAV vector of the first rAAV particle isencapsidated by an Anc80 capsid. In some embodiments the recombinant AAVvector of the second rAAV particle is encapsidated by an Anc80 capsid.In some embodiments the recombinant AAV vectors of the first and secondrAAV particles are each independently encapsidated by an Anc80 capsid.In some embodiment, the Anc80 capsid comprises a polypeptide of SEQ IDNO: 109.

In one aspect the present disclosure provides a composition comprising(a) a first rAAV particle comprising a recombinant AAV vector thatcomprises, in order of 5′ to 3′: a 5′ ITR sequence of SEQ ID NO: 97; aCAG promoter comprising a CMV early enhancer element of SEQ ID NO: 98, achicken beta actin gene sequence of SEQ ID NO: 99, and a chimeric intronof SEQ ID NO: 100; a 5′OTOF coding region that comprises exons 1 to (andthrough) 21 of OTOF cDNA; a SD intron sequence of SEQ ID NO: 102; an AKrecombinogenic sequence of SEQ ID NO: 103; and a 3′ ITR sequence of SEQID NO: 104; and (b) a second rAAV particle comprising a recombinant AAVvector that comprises a 5′ ITR sequence of SEQ ID NO: 97; an AKrecombinogenic sequence of SEQ ID NO: 103; a SA intron sequence of SEQID NO: 106; a 3′ OTOF coding region that comprises exons 22 to (andthrough) exon 48 of OTOF cDNA; a bgH polyA sequence of SEQ ID NO: 108;and a 3′ ITR sequence of SEQ ID NO: 104. In some embodiments, the 5′OTOFcoding region is SEQ ID NO: 101. In some embodiments, the 5′OTOF codingregion is at least 70% identical to SEQ ID NO: 101, and encodes the sameamino acid sequence as SEQ ID NO: 101. In some embodiments, the 3′OTOFcoding region is SEQ ID NO: 107. In some embodiments, the 3′OTOF codingregion is at least 70%, at least 80%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or100% identical to SEQ ID NO: 107, and encodes the same amino acidsequence as encoded by SEQ ID NO: 107. In some embodiments therecombinant AAV vectors in the first and second rAAV particles are eachindependently encapsidated by an Anc80 capsid. In some embodiment, theAnc80 capsid comprises a polypeptide of SEQ ID NO: 109.

In one aspect when the aforementioned composition is introduced into ahuman cell the recombinant AAV vectors of the first and second rAAVparticles undergo concatamerization or homologous recombination witheach other, thereby forming a recombined nucleic acid that encodes afull-length otoferlin protein within the cell.

In one aspect the present disclosure provides a method comprisingintroducing into a cochlea of a mammal (e.g., a human) a therapeuticallyeffective amount of any of the aforementioned composition. In someembodiments, the mammal has been previously identified as having adefective otoferlin gene.

In one aspect the present disclosure provides a method of increasingexpression of a full-length otoferlin protein in a mammalian cell, themethod comprising introducing any of the aforementioned compositionsinto the mammalian cell, e.g., an inner hair cell, e.g., a human cell.In some embodiments, the mammalian cell has previously been determinedto have a defective otoferlin gene.

In one aspect the present disclosure provides a method of increasingexpression of a full-length otoferlin protein in an inner hair cell in acochlea of a mammal, e.g., a human, the method comprising introducinginto the cochlea of the mammal a therapeutically effective amount of anyof the aforementioned compositions. In some embodiments, the mammal hasbeen previously identified as having a defective otoferlin gene.

In one aspect the present disclosure provides a method of treatingnon-symptomatic sensorineural hearing loss in a subject, e.g., a humanidentified as having a defective otoferlin gene, the method comprisingadministering a therapeutically effective amount of any of theaforementioned compositions into the cochlea of the subject. In someembodiments, the method further comprises, prior to the administeringstep, determining that the subject has a defective otoferlin gene. Insome embodiments the composition is administered to the cochlea using amicrocatheter. In some embodiments the microcatheter is shaped such thatit can enter the middle ear cavity via the external auditory canal andcontact the end of the microcatheter with the RWM. In some embodimentsthe distal end of the microcatheter is comprised of at least onemicroneedle with diameter of between 10 and 1,000 microns.

In one aspect the present disclosure provides a kit comprising any ofthe aforementioned compositions. In some embodiments the composition ispre-loaded in a device, e.g., a microcatheter. In some embodiments themicrocatheter is shaped such that it can enter the middle ear cavity viathe external auditory canal and contact the end of the microcatheterwith the RWM. In some embodiments the distal end of the microcatheter iscomprised of at least one microneedle with diameter of between 10 and1,000 microns.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

DETAILED DESCRIPTION

Mutations in OTOF lead to DFNB9, a recessively inherited, non-syndromicprelingual hearing disorder. Deficiency in otoferlin, the proteinencoded by OTOF, abolishes fast exocytosis from auditory inner haircells (IHCs). Due the impairment of neurotransmission at the firstauditory synapse, no sound signals are transmitted to the brain,explaining the profound deafness.

The presently claimed methods were discovered to result in expression offull-length otoferlin in inner auditory hair cells and to successfullyrestore hearing with ABR thresholds of 30 dB to 70 dB for click stimuliand 50 dB to 90 dB for pure tones in otoferlin knock-out mice. In viewof this discovery, provided herein are compositions and methods fortreating non-symptomatic sensorineural hearing loss in a subject (e.g.,a human) identified as having a defective otoferlin gene.

Provided herein are compositions that include at least two differentnucleic acid vectors, where: each of the at least two different vectorsincludes a coding sequence that encodes a different portion of anotoferlin protein, each of the encoded portions being at least 30 aminoacid residues in length, where the amino acid sequence of each of theencoded portions may optionally partially overlap with the amino acidsequence of a different one of the encoded portions; no single vector ofthe at least two different vectors encodes an active otoferlin protein(e.g., a full-length otoferlin protein); at least one of the codingsequences comprises a nucleotide sequence spanning two neighboring exonsof otoferlin genomic DNA, and lacks an intronic sequence between the twoneighboring exons; and, when introduced into a mammalian cell, the atleast two different vectors undergo homologous recombination with eachother, thereby forming a recombined nucleic acid, where the recombinednucleic acid encodes an active otoferlin protein (e.g., a full-lengthotoferlin protein). In some examples, the recombined nucleic acid thatencodes an active otoferlin protein (e.g., a full-length otoferlinprotein) exists as an episome in a mammalian cell (e.g., any of thetypes of mammalian cells described herein). Also provided are kits thatinclude any of the compositions described herein. Also provided hereinare methods that include introducing into a cochlea of a mammal atherapeutically effective amount of any of the compositions describedherein.

Also provided herein are methods of increasing expression of an activeotoferlin protein (e.g., a full-length otoferlin protein) in a mammaliancell that include introducing any of the compositions described hereininto the mammalian cell. Also provided herein are methods of increasingexpression of an active otoferlin protein (e.g., a full-length otoferlinprotein) in an inner hair cell in a cochlea of a mammal that includeintroducing into the cochlea of the mammal a therapeutically effectiveamount of any of the compositions described herein. Also provided hereinare methods of treating non-symptomatic sensorineural hearing loss in asubject identified as having a defective otoferlin gene that include:administering a therapeutically effective amount of any of thecompositions described herein into the cochlea of the subject.

Additional non-limiting aspects of the compositions, kits, and methodsare described herein and can be used in any combination withoutlimitation.

Otoferlin

The human OTOF gene encodes otoferlin, which is a protein that, in someembodiments, plays a critical role in priming, fusion, and/orreplenishing of synaptic vesicles of inner hair cell synapses duringsound encoding. To date, several hundred mutations in the human OTOFgene have been identified to cause profound prelingual deafness DFNB9.Such mutations are the cause of deafness in 2-8% of people that are bornwith an autosomal, recessively inherited, non-syndromic deafness indifferent populations (Rodriguez-Ballesteros et al. (2008) Hum. Mutat.29 823-831; Choi et al. (2009) Clinical Genetics 75 237-243; Duman etal. (2011) Genet Test Mol Biomarkers 15 29-33; Varga et al. (2006) J MedGenet 43 576-581; Iwasa et al. (2013) BMC Med. Genet. 14 95). Biallelicotoferlin gene mutations cause localized, synaptic transmission defectsbetween hair cells and the auditory nerve. Otoferlin enables sensorycells to release neurotransmitters in response to stimulation by soundto activate auditory neurons and those neurons carry electronicallyencoded acousting information to the brain to produce “hearing.” Whenbiallelic mutations in OTOF are present, that transmission is impairedand, as a result, a majority of subjects have congenital, severe toprofound sensorineural hearing loss. For example, two substitutions inexon 15 at positions 490 and 515 in the conserved C2C domain ofotoferlin cause DFNB9 (Mirqhomizadeh et al. (2002) Neurobiol. Dis.10(2): 157-164). Migliosi et al. found a novel mutation Q829X in OTOF inSpanish subjects with prelingual non-syndromic hearing loss (Migliosi etal. (2002) J. Med. Genet. 39(7): 502-506).

Additional exemplary mutations in a otoferlin gene detected in subjectshaving hearing loss and methods of sequencing a nucleic acid encodingotoferlin are described in, e.g., Rodriguez-Ballesteros et al. (2003)Hum Mutat. 22: 451-456; Wang et al. (2010) BMC Med Genet. 11:79;Yildirim-Baylan et al. (2014) Int. J. Pediatr. Otorhinolaryngol 78:950-953; Choi et al. (2009) Clin. Genet. 75(3): 237-243; and Marlin etal. (2010) Biochem Biophys Res Commun 394: 737-742.

Otoacoustic emissions from DFNB9 subjects are normal, at least for thefirst decade of life, indicating morphological integrity of the innerear and proper function of outer hair cells. Apart from the lack ofsynaptic transmission and the subsequent loss of synapses, themorphology and physiology of the inner ear remains preserved in DFNB9,at least during the first decade in life in humans. Accordingly, in someembodiments, restoration of OTOF and/or otoferlin function may serve tomitigate or prevent secondary degeneration of one or more cochlearstructures.

Studies in mouse models revealed that synapses are structurally normal,and IHCs preserve normal synapse numbers in mice within the firstpostnatal week. Between P6 and P15, about half of the synapses get lost(Roux et al. (2006) Cell 127 277-289). Animal models allowed studyingthe effect of mutations in otoferlin on synaptic transmission byrecording the change in plasma membrane capacitance after vesicle fusionand the activity in the auditory nerve. In otoferlin knock-out (Otof−/−)mice, almost no exocytosis could be triggered in IHCs by depolarizationinduced Ca2+ influx through voltage gated Ca2+ channels (Roux et al.(2006) Cell 127 277-289). In profoundly hearing impaired pachanga(OtofPga/Pga) mice with a random point mutation in the C2F domain, short(<10 ms) depolarizations of the IHCs elicited vesicle fusion of similarsize as in wild type mice, however sustained stimulations uncovered astrong deficiency in replenishing vesicles to the readily releasablepool (Pangrsic et al. (2010) Nat. Neurosci. 13 869-876). The p.Ile515Thrmutation, found in human subjects with only mildly elevated hearingthresholds but a severe reduction in speech understanding and atemperature-dependent deafening (Varga et al. (2006) J Med Genet 43576-581), uncovered an intermediate phenotype when studied in a mousemodel (Strenzke et al. (2016) EMBO J. 35:2519-2535). TheseOtofI515T/I515T mice showed a moderate elevation of hearing thresholdswhen assessed by ABR with a reduction in wave I amplitude, but normalauditory thresholds in behavioral tests and recordings of singleauditory nerve units. Exocytosis of the RRP is again intact, butsustained exocytosis is reduced, although not as severe as inOtofPga/Pga. While in wild type mice at room temperature, during asustained stimulus 750 vesicles can fuse per second at each active zone,this rate drops to 350 vesicles/s/active zone in OtofJ515T/I515T miceand to 200 vesicles/s/active zone in OtofPga/Pga IHCs (Pangrsic et al.(2010) Nat. Neurosci. 13 869-876; Strenzke et al. (2016) EMBO J. 352519-2535). This correlated with lower otoferlin protein levels at theplasma membrane of IHCs, indicating that the amount of otoferlin scaleswith exocytosis and hearing (Strenzke et al. (2016) EMBO J. 352519-2535).

Methods of detecting mutations in a gene are well-known in the art.Non-limiting examples of such techniques include: real-time polymerasechain reaction (RT-PCR), PCR, sequencing, Southern blotting, andNorthern blotting.

The OTOF gene encodes otoferlin, a protein that is involved in synapticvesicle exocytosis in cochlear hair cells (see, e.g., Johnson andChapman (2010) J. Cell Biol. 191(1):187-198; and Heidrych et al. (2008)Hum. Mol. Genet. 17:3814-3821).

The human OTOF gene is located on chromosome 2p23.3. It contains 48exons encompassing ˜132 kilobases (kb) (NCBI Accession No. NG009937.1).The mRNA encoding the long-form of otoferlin expressed in the brainincludes 48 exons (Yasunaga et al., Am. J. Hum. Genet. 67:591-600,2000). Forward and reverse primers that can be used to amplify each ofthe 48 exons in the OTOF gene are described in Table 2 of Yasunaga etal., Am. J. Hum. Genet. 67:591-600, 2000. In some examples, thefull-length OTOF protein is a full-length wildtype OTOF protein. Thefull-length wildtype OTOF protein expressed from the human OTOF gene is1997 residues in length.

An exemplary human wildtype otoferlin protein is or includes thesequence of any one of SEQ ID NOs: 1-5. Isoform e of human otoferlinprotein (SEQ ID NO: 5) is encoded by an mRNA that includes exon 48 anddoes not include exon 47 of the otoferlin gene (Yasunaga et al., Am. J.Hum. Genet. 67:591-600, 2000). In some embodiments, the active otoferlinprotein has the sequence of SEQ ID NO: 5, but is missing the 20 aminoacids including the RXR motif identified in Strenzke et al., EMBO J.35(23):2499-2615, 2016. Non-limiting examples of nucleic acids encodinga wildtype otoferlin protein are or include any one of SEQ ID NO: 7-11.As can be appreciated in the art, at least some or all of the codons inSEQ ID NO: 7-11 can be codon-optimized to allow for optimal expressionin a non-human mammal or in a human. Orthologs of human otoferlinproteins are known in the art.

Human Otoferlin Protein cDNA Sequences:

Human canonical (long) isoform sequence (otoferlin protein) (SEQ IDNO: 1) (also called otoferlin isoform a) (NCBI Accession No. AAD26117.1)

Human Isoform 2 (short 1) (otoferlin protein) (SEQ ID NO: 2) (alsocalled otoferlin isoform d) (NCBI Accession No. NP_919304.1)

Human Isoform 3 (short 2) (otoferlin protein) (SEQ ID NO: 3) (alsocalled otoferlin isofom c) (NCBI Accession No. NP_919303.1)

Human Isoform 4 (short 3) (otoferlin protein) (SEQ ID NO: 4) (alsocalled otoferlin isoform b) (NCBI Accession No. NP_004793.2)

Human Isoform 5 (short 4) (otoferlin protein) (SEQ ID NO: 5) (alsocalled otoferlin isoform e) (NCBI Accession No. NP_001274418.1)

Complete cds (otoferlin cDNA) (www.ncbi.nlm.nih.gov/nuccore/AF107403.1)(SEQ ID NO: 6) (encodes the protein of SEQ ID NO: 1)

Human Otoferlin Transcript Variant 1(www.ncbi.nlm.nih.gov/nuccore/NM_194248.2) (SEQ ID NO: 7) (encodes theprotein of SEQ ID NO: 1)

Human Otoferlin Transcript Variant 2(www.ncbi.nlm.nih.gov/nuccore/NM_004802.3) (SEQ ID NO: 8) (encodes theprotein of SEQ ID NO: 4)

Human Otoferlin Transcript Variant 3(www.ncbi.nlm.nih.gov/nuccore/NM_194322.2) (SEQ ID NO: 9) (encodes theprotein of SEQ ID NO: 3)

Human Otoferlin Transcript Variant 4(www.ncbi.nlm.nih.gov/nuccore/NM_194323.2) (SEQ ID NO: 10) (encodes theprotein of SEQ ID NO: 2)

Human Otoferlin Transcript Variant 5(www.ncbi.nlm.nih.gov/nuccore/NM_001287489.1) (SEQ ID NO: 11) (encodesthe protein of SEQ ID NO: 5)

A non-limiting example of a human wildtype otoferlin genomic DNAsequence is SEQ ID NO: 12. The exons in SEQ ID NO: 12 are: nucleotidepositions 5001-5206 (exon 1), nucleotide positions 25925-25983 (exon 2),nucleotide positions 35779-35867 (exon 3), nucleotide positions44590-44689 (exon 4), nucleotide positions 47100-47281 (exon 5),nucleotide positions 59854-59927 (exon 6), nucleotide positions61273-61399 (exon 7), nucleotide positions 61891-61945 (exon 8),nucleotide positions 68626-68757(exon 9), nucleotide positions73959-74021 (exon 10), nucleotide positions 74404-74488 (exon 11),nucleotide positions 79066-79225 (exon 12), nucleotide positions80051-80237 (exon 13), nucleotide positions 81107-81293 (exon 14),nucleotide positions 82690-82913 (exon 15), nucleotide positions83388-83496 (exon 16), nucleotide positions 84046-84226 (exon 17),nucleotide positions 84315-84435 (exon 18), nucleotide positions85950-86050 (exon 19), nucleotide positions 86193-86283 (exon 20),nucleotide positions 86411-86527 (exon 21), nucleotide positions86656-86808 (exon 22), nucleotide positions 87382-87571 (exon 23),nucleotide positions 87661-87785 (exon 24), nucleotide positions88206-88340 (exon 25), nucleotide positions 89025-89186 (exon 26),nucleotide positions 89589-89708 (exon 27), nucleotide positions90132-90293 (exon 28), nucleotide positions 90405-90567 (exon 29),nucleotide positions 91050-91180 (exon 30), nucleotide positions92549-92578 (exon 31), nucleotide positions 92978-93106 (exon 32),nucleotide positions 95225-95291 (exon 33), nucleotide positions96198-96334 (exon 34), nucleotide positions 96466-96600 (exon 35),nucleotide positions 96848-96985 (exon 36), nucleotide positions97623-97750 (exon 37), nucleotide positions 97857-98027 (exon 38),nucleotide positions 98670-98830 (exon 39), nucleotide positions99593-99735 (exon 40), nucleotide positions 100128-100216 (exon 41),nucleotide positions 101518-101616 (exon 42), nucleotide positions101762-102003 (exon 43), nucleotide positions 102669-102847 (exon 44),nucleotide positions 102952-103052 (exon 45), nucleotide positions103494-103691 (exon 46), nucleotide positions 105479-106496 (exon 47),and exon 48 (the sequence starting with CCGGCCCGAC; see also thedescription of this exon in Yasunaga et al., Am. J. Hum. Genet.67:591-600, 2000).

The introns are located between each contiguous pair of exons in SEQ IDNO: 12, i.e., at nucleotide positions 100-5001 (intron 1), nucleotide5207-25924 (intron 2), nucleotide positions 25984-35778 (intron 3),nucleotide positions 35868-44589 (intron 4), nucleotide positions44690-47099 (intron 5), nucleotide positions 47282-59853(intron 6),nucleotide positions 59928-61272 (intron 7), nucleotide positions61400-61890 (intron 8), nucleotide positions 61946-68625 (intron 9),nucleotide positions 68758-73958 (intron 10), nucleotide positions74022-74403 (intron 11), nucleotide positions 74489-79065 (intron 12),nucleotide positions 79226-80050 (intron 13), nucleotide positions80238-81106 (intron 14), nucleotide positions 81294-82689 (intron 15),nucleotide positions 82914-83387 (intron 16), nucleotide positions83497-84045 (intron 17), nucleotide positions 84227-84314 (intron 18),nucleotide positions 84436-85949 (intron 19), nucleotide positions86051-86192 (intron 20), nucleotide positions 86284-86410 (intron 21),nucleotide positions 86528-86655 (intron 22), nucleotide positions86809-87381 (intron 23), nucleotide positions 87572-87660 (intron 24),nucleotide positions 87786-88205 (intron 25), nucleotide positions88341-89024 (intron 26), nucleotide positions 89187-89588 (intron 27),nucleotide positions 89709-90131 (intron 28), nucleotide positions90294-90404 (intron 29), nucleotide positions 90568-91049 (intron 30),nucleotide positions 91181-92548 (intron 31), nucleotide positions92579-92977 (intron 32), nucleotide positions 93107-95224 (intron 33),nucleotide positions 95292-96197 (intron 34), nucleotide positions96335-96465 (intron 35), nucleotide positions 96601-96847 (intron 36),nucleotide positions 96986-97622 (intron 37), nucleotide positions97751-97856 (intron 38), nucleotide positions 98028-98669 (intron 39),nucleotide positions 98831-99592 (intron 40), nucleotide positions99736-100127 (intron 41), nucleotide positions 100217-101517 (intron42), nucleotide positions 101617-101761 (intron 43), nucleotidepositions 102004-102668 (intron 44), nucleotide positions 102848-102951(intron 45), nucleotide positions 103053-103494 (intron 46), nucleotidepositions 103692-105478 (intron 47), and nucleotide positions106497-108496 (intron 48).

In some embodiments, an otoferlin gene may be split into two or moresegments between or within any appropriate exons and/or introns, whereeach segment is included in a different vector of the presentdisclosure. In some embodiments, the otoferlin gene is split at exon 21,i.e., with exons 1 to (and through) 21 in a first vector and exons 22 to(and through) exon 48 in a second vector. In some such embodiments theotoferlin segments in the first and second vectors are derived from anotoferlin cDNA sequence and lack introns, i.e., with exons 1 to (andthrough) 21 in a first vector and exons 22 to (and through) exon 48 in asecond vector, each vector lacking otoferlin introns. In someembodiments, an otoferlin gene may be split at one or more other exonsand/or introns as long as, when combined with all other components of avector, the packaging capacity of the vector is not exceeded.

Human Otoferlin Gene Sequence (ncbi.nlm.nih.gov/nuccore/224465243) (SEQID NO: 12)

Mouse Otoferlin Protein (SEQ ID NO: 13) (NCBI Accession No.NP_001300696.1)

Mouse Otoferlin cDNA (SEQ ID NO: 14) (NCBI Accession No. NM_001313767.1)

Mouse Otoferlin Gene Sequence (www.ncbi.nlm.nih.gov/gene/83762) (SEQ IDNO: 15) Accession: NC_000071 REGION: complement(30367066 . . . 30462730)GPC_000000778; NCBI Reference Sequence: NC_000071.6

Zebrafish Otoferlin A Gene Sequence (www.ncbi.nlm.nih.gov/gene/557476)(SEQ ID NO: 16) ACCESSION NC_007131 REGION: 31173357.31310109GPC_000001574 NCBI Reference Sequence: NC 007131.7

Rhesus Monkey Otoferlin Gene Sequence (www.ncbi.nlm.nih.gov/gene/696717)(SEQ ID NO: 17) ACCESSION NC_027905 REGION: complement(26723411 . . .26826586) GPC_000002105 NCBI Reference Sequence: NC_027905.1

Dog Otoferlin Gene Sequence (www.ncbi.nlm.nih.gov/gene/607961) (SEQ IDNO: 18) ACCESSION NC_006599 REGION: complement(20518502 . . . 20619461)GPC_000000676 NCBI Reference Sequence: NC_006599.3

Chimpanzee Otoferlin Gene Sequence (www.ncbi.nlm.nih.gov/gene/459083)(SEQ ID NO: 19) ACCESSION NC_006469 REGION: complement(27006052 . . .27107747) GPC_000002338 NCBI Reference Sequence: NC_006469.4

Rat Otoferlin Protein (SEQ ID NO: 20)

Zebrafish Otoferlin Protein (SEQ ID NO: 21)

Cow Otoferlin Protein (SEQ ID NO: 22)

Baboon Otoferlin Protein (SEQ ID NO: 23)

In some embodiments, a first vector comprises a 5′ portion of OTOF cDNA,e.g., as shown in SEQ ID NO: 94. In some embodiments, a second vectorcomprises a 3′ portion of OTOF cDNA, e.g., as shown in SEQ ID NO: 95.

5′mOTOF DNA Sequence (SEQ ID NO: 94)ATGGCCCTGATTGTTCACCTCAAGACTGTCTCAGAGCTCCGAGGCAAAGGTGACCGGATTGCCAAAGTCACTTTCCGAGGGCAGTCTTTCTACTCCCGGGTCCTGGAGAACTGCGAGGGTGTGGCTGACTTTGATGAGACGTTCCGGTGGCCAGTGGCCAGCAGCATCGACCGGAATGAAGTGTTGGAGATTCAGATTTTCAACTACAGCAAAGTCTTCAGCAACAAGCTGATAGGGACCTTCTGCATGGTGCTGCAGAAAGTGGTGGAGGAGAATCGGGTAGAGGTGACCGACACGCTGATGGATGACAGCAATGCTATCATCAAGACCAGCCTGAGCATGGAGGTCCGGTATCAGGCCACAGATGGCACTGTGGGCCCCTGGGATGATGGAGACTTCCTGGGAGATGAATCCCTCCAGGAGGAGAAGGACAGCCAGGAGACAGATGGGCTGCTACCTGGTTCCCGACCCAGCACCCGGATATCTGGCGAGAAGAGCTTTCGCAGAGCGGGAAGGAGTGTGTTCTCGGCCATGAAACTCGGCAAAACTCGGTCCCACAAAGAGGAGCCCCAAAGACAAGATGAGCCAGCAGTGCTGGAGATGGAGGACCTGGACCACCTAGCCATTCAGCTGGGGGATGGGCTGGACCCTGACTCCGTGTCTCTAGCCTCGGTCACCGCTCTCACCAGCAATGTCTCCAACAAACGGTCTAAGCCAGATATTAAGATGGAGCCCAGTGCTGGAAGGCCCATGGATTACCAGGTCAGCATCACAGTGATTGAGGCTCGGCAGCTGGTGGGCTTGAACATGGACCCTGTGGTGTGTGTGGAGGTGGGTGATGACAAGAAATACACGTCAATGAAGGAGTCCACAAACTGCCCTTACTACAACGAGTACTTTGTCTTCGACTTCCATGTCTCTCCTGATGTCATGTTTGACAAGATCATCAAGATCTCGGTTATCCATTCTAAGAACCTGCTTCGGAGCGGCACCCTGGTGGGTTCCTTCAAAATGGATGTGGGGACTGTGTATTCCCAGCCTGAACACCAGTTCCATCACAAATGGGCCATCCTGTCAGACCCCGATGACATCTCTGCTGGGTTGAAGGGTTATGTAAAGTGTGATGTCGCTGTGGTGGGCAAGGGAGACAACATCAAGACACCCCACAAGGCCAACGAGACGGATGAGGACGACATTGAAGGGAACTTGCTGCTCCCCGAGGGCGTGCCCCCCGAACGGCAGTGGGCACGGTTCTATGTGAAAATTTACCGAGCAGAGGGACTGCCCCGGATGAACACAAGCCTCATGGCCAACGTGAAGAAGGCGTTCATCGGTGAGAACAAGGACCTCGTCGACCCCTATGTGCAAGTCTTCTTTGCTGGACAAAAGGGCAAAACATCAGTGCAGAAGAGCAGCTATGAGCCGCTATGGAATGAGCAGGTCGTCTTCACAGACTTGTTCCCCCCACTCTGCAAACGCATGAAGGTGCAGATCCGGGACTCTGACAAGGTCAATGATGTGGCCATCGGCACCCACTTCATCGACCTGCGCAAGATTTCCAACGATGGAGACAAAGGCTTCCTGCCTACCCTCGGTCCAGCCTGGGTGAACATGTACGGCTCCACGCGCAACTACACACTGCTGGACGAGCACCAGGACTTGAATGAAGGCCTGGGGGAGGGTGTGTCCTTCCGGGCCCGCCTCATGTTGGGACTAGCTGTGGAGATCCTGGACACCTCCAACCCAGAGCTCACCAGCTCCACGGAGGTGCAGGTGGAGCAGGCCACGCCTGTCTCGGAGAGCTGCACAGGGAGAATGGAAGAATTTTTTCTATTTGGAGCCTTCTTGGAAGCCTCAATGATTGACCGGAAAAATGGGGACAAGCCAATTACCTTTGAGGTGACCATAGGAAACTACGGCAATGAAGTCGATGGTATGTCCCGGCCCCTGAGGCCTCGGCCCCGGAAAGAGCCTGGGGATGAAGAAGAGGTAGACCTGATTCAGAACTCCAGTGACGATGAAGGTGACGAAGCCGGGGACCTGGCCTCGGTGTCCTCCACCCCACCTATGCGGCCCCAGATCACGGACAGGAACTATTTCCACCTGCCCTACCTGGAGCGCAAGCCCTGCATCTATATCAAGAGCTGGTGGCCTGACCAGAGGCGGCGCCTCTACAATGCCAACATCATGGATCACATTGCTGACAAGCTGGAAGAAGGCCTGAATGATGTACAGGAGATGATCAAAACGGAGAAGTCCTACCCGGAGCGCCGCCTGCGGGGTGTGCTAGAGGAACTCAGCTGTGGCTGCCACCGCTTCCTCTCCCTCTCGGACAAGGACCAGGGCCGCTCGTCCCGCACCAGGCTGGATCGAGAGCGTCTTAAGTCCTGTATGAGGGAGTTGGAGAGCATGGGACAGCAGGCCAAGAGCCTGAGGGCTCAGGTGAAGCGGCACACTGTTCGGGACAAGCTGAGGTCATGCCAGAACTTTCTGCAGAAGCTACGCTTCCTGGCGGATGAG 3′mOTOF DNA Sequence(SEQ ID NO: 95) CCCCAGCACAGCATTCCGGATGTGTTCATTTGGATGATGAGCAACAACAAACGTATCGCCTATGCCCGCGTGCCTTCCAAAGACCTGCTCTTCTCCATCGTGGAGGAGGAACTGGGCAAGGACTGCGCCAAAGTCAAGACCCTCTTCCTGAAGCTGCCAGGGAAGAGGGGCTTCGGCTCGGCAGGCTGGACAGTACAGGCCAAGCTGGAGCTCTACCTGTGGCTGGGCCTCAGCAAGCAGCGAAAGGACTTCCTGTGTGGTCTGCCCTGTGGCTTCGAGGAGGTCAAGGCAGCCCAAGGCCTGGGCCTGCATTCCTTTCCGCCCATCAGCCTAGTCTACACCAAGAAGCAAGCCTTCCAGCTCCGAGCACACATGTATCAGGCCCGAAGCCTCTTTGCTGCTGACAGCAGTGGGCTCTCTGATCCCTTTGCCCGTGTCTTCTTCATCAACCAGAGCCAATGCACTGAGGTTCTAAACGAGACACTGTGTCCCACCTGGGACCAGATGCTGGTATTTGACAACCTGGAGCTGTACGGTGAAGCTCACGAGTTACGAGATGATCCCCCCATCATTGTCATTGAAATCTACGACCAGGACAGCATGGGCAAAGCCGACTTCATGGGCCGGACCTTCGCCAAGCCCCTGGTGAAGATGGCAGATGAAGCATACTGCCCACCTCGCTTCCCGCCGCAGCTTGAGTACTACCAGATCTACCGAGGCAGTGCCACTGCCGGAGACCTACTGGCTGCCTTCGAGCTGCTGCAGATTGGGCCATCAGGGAAGGCTGACCTGCCACCCATCAATGGCCCAGTGGACATGGACAGAGGGCCCATCATGCCTGTGCCCGTGGGAATCCGGCCAGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTGAGGGACCTAAAGAGGGTGAACCTGGCCCAGGTGGACCGACCACGGGTGGACATCGAGTGTGCAGGAAAGGGGGTACAATCCTCCCTGATTCACAATTATAAGAAGAACCCCAACTTCAACACGCTGGTCAAGTGGTTTGAAGTGGACCTCCCGGAGAATGAGCTCCTGCACCCACCCTTGAACATCCGAGTGGTAGATTGCCGGGCCTTTGGACGATACACCCTGGTGGGTTCCCACGCAGTCAGCTCACTGAGGCGCTTCATCTACCGACCTCCAGACCGCTCAGCCCCCAACTGGAACACCACAGGGGAGGTTGTAGTAAGCATGGAGCCTGAGGAGCCAGTTAAGAAGCTGGAGACCATGGTGAAACTGGATGCGACTTCTGATGCTGTGGTCAAGGTGGATGTGGCTGAAGATGAGAAGGAAAGGAAGAAGAAGAAAAAGAAAGGCCCGTCAGAGGAGCCAGAGGAGGAAGAGCCCGATGAGAGCATGCTGGATTGGTGGTCCAAGTACTTCGCCTCCATCGACACAATGAAGGAGCAACTTCGACAACATGAGACCTCTGGAACTGACTTGGAAGAGAAGGAAGAGATGGAAAGCGCTGAGGGCCTGAAGGGACCAATGAAGAGCAAGGAGAAGTCCAGAGCTGCAAAGGAGGAGAAAAAGAAGAAAAACCAGAGCCCTGGCCCTGGCCAGGGATCGGAGGCTCCTGAGAAGAAGAAAGCCAAGATCGATGAGCTTAAGGTGTACCCCAAGGAGCTGGAATCGGAGTTTGACAGCTTTGAGGACTGGCTGCACACCTTCAACCTGTTGAGGGGCAAGACGGGAGATGATGAGGATGGCTCCACAGAGGAGGAGCGCATAGTAGGCCGATTCAAGGGCTCCCTCTGTGTGTACAAAGTGCCACTCCCAGAAGATGTATCTCGAGAAGCTGGCTATGATCCCACCTATGGAATGTTCCAGGGCATCCCAAGCAATGACCCCATCAATGTGCTGGTCCGAATCTATGTGGTCCGGGCCACAGACCTGCACCCGGCCGACATCAATGGCAAAGCTGACCCCTATATTGCCATCAAGTTAGGCAAGACCGACATCCGAGACAAGGAGAACTACATCTCCAAGCAGCTCAACCCTGTGTTTGGGAAGTCCTTTGACATTGAGGCCTCCTTCCCCATGGAGTCCATGTTGACAGTGGCCGTGTACGACTGGGATCTGGTGGGCACTGATGACCTCATCGGAGAAACCAAGATTGACCTGGAAAACCGCTTCTACAGCAAGCATCGCGCCACCTGCGGCATCGCACAGACCTATTCCATACATGGCTACAATATCTGGAGGGACCCCATGAAGCCCAGCCAGATCCTGACACGCCTCTGTAAAGAGGGCAAAGTGGACGGCCCCCACTTTGGTCCCCATGGGAGAGTGAGGGTTGCCAACCGTGTCTTCACGGGGCCTTCAGAAATAGAGGATGAGAATGGTCAGAGGAAGCCCACAGATGAGCACGTGGCACTGTCTGCTCTGAGACACTGGGAGGACATCCCCCGGGTGGGCTGCCGCCTTGTGCCGGAACACGTGGAGACCAGGCCGCTGCTCAACCCTGACAAGCCAGGCATTGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACACCTCTGGATATATCCCCCAGGAAACCCAAGAAGTACGAGCTGCGGGTCATCGTGTGGAACACAGACGAGGTGGTCCTGGAAGACGATGATTTCTTCACGGGAGAGAAGTCCAGTGACATTTTTGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAACAGGACACAGATGTCCACTATCACTCCCTCACGGGGGAGGGCAACTTCAACTGGAGATACCTCTTCCCCTTCGACTACCTAGCGGCCGAAGAGAAGATCGTTATGTCCAAAAAGGAGTCTATGTTCTCCTGGGATGAGACGGAGTACAAGATCCCTGCGCGGCTCACCCTGCAGATCTGGGACGCTGACCACTTCTCGGCTGACGACTTCCTGGGGGCTATCGAGCTGGACCTGAACCGGTTCCCGAGGGGCGCTAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTACCCCTGGTTTCCATCTTTAAACAGAAACGTGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAATGATGAGTTTGAGCTCACAGGCAAAGTGGAGGCGGAGCTACACCTACTCACGGCAGAGGAGGCAGAGAAGAACCCTGTGGGCCTGGCTCGCAATGAACCTGATCCCCTAGAAAAACCCAACCGGCCTGACACGGCATTCGTCTGGTTCCTGAACCCACTCAAATCTATCAAGTACCTCATCTGCACCCGGTACAAGTGGCTGATCATCAAGATCGTGCTGGCGCTGCTGGGGCTGCTCATGCTGGCCCTCTTCCTTTACAGCCTCCCAGGCTACATGGTCAAGAAGCTCCTAGGGGCCTGA

Some embodiments of any of the compositions described herein can includea first vector including the coding sequence of SEQ ID NO: 94 (orinclude a sequence that is at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, or at least 99% identical to SEQ ID NO: 94).Some embodiments of any of the compositions described herein can includea second vector including the coding sequence of SEQ ID NO: 95 (orinclude a sequence that is at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, or at least 99% identical to SEQ ID NO: 95).

Some embodiments of any of the compositions described herein can includea first vector with a 5′ OTOF coding region that comprises exons 1 to(and through) 21 of OTOF cDNA. Some embodiments of any of thecompositions described herein can include a first vector that comprisesthe nucleotide sequence of SEQ ID NO: 101 (or a sequence that is atleast 70%, at least 75%, at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, or at least 99% identical to the nucleotidesequence of SEQ ID NO: 101). Some embodiments of any of the compositionsdescribed herein can include a first vector that comprises a nucleotidesequence that is at least 70%, at least 75%, at least 80%, at least 82%,at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, or at least 99% identical to thenucleotide sequence of SEQ ID NO: 101, and encodes the same amino acidsequence as SEQ ID NO: 101. In some embodiments, a composition of thepresent disclosure includes a first vector that comprises a codonoptimized version of SEQ ID NO: 101, i.e., a nucleotide sequence thatencodes the same amino acid sequence as SEQ ID NO: 101 but with codonsthat have been optimized for expression in a particular cell type, e.g.,a mammalian cell, e.g., a human cell. In some embodiments the firstvector does not include any other portion of an OTOF gene. In someembodiments the first vector does not include any other portion of OTOFcDNA.

Some embodiments of any of the compositions described herein can includea second vector with a 3′ OTOF coding region that comprises exons 22 to(and through) exon 48 of OTOF cDNA. Some embodiments of any of thecompositions described herein can include a second vector that comprisesthe nucleotide sequence of SEQ ID NO: 108 (or a sequence that is atleast 70%, at least 75%, at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, or at least 99% identical to the nucleotidesequence of SEQ ID NO: 108). Some embodiments of any of the compositionsdescribed herein can include a second vector that comprises a nucleotidesequence that is at least 70%, at least 75%, at least 80%, at least 82%,at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, or at least 99% identical to thenucleotide sequence of SEQ ID NO: 108, and encodes the same amino acidsequence as SEQ ID NO: 108. In some embodiments, a composition of thepresent disclosure includes a second vector that comprises a codonoptimized version of SEQ ID NO: 108, i.e., a nucleotide sequence thatencodes the same amino acid sequence as SEQ ID NO: 108 but with codonsthat have been optimized for expression in a particular cell type, e.g.,a mammalian cell, e.g., a human cell. In some embodiments the secondvector does not include any other portion of an OTOF gene. In someembodiments the second vector does not include any other portion of OTOFcDNA.

Vectors

Provided herein are compositions of matter and methods of use for thetreatment of a disease such as nonsyndromic hearing loss, using nucleicacid therapeutics such as auditory polypeptide messenger RNAs.Preferably, the auditory polypeptide nucleic acids are present in viralvectors, such as adeno-associated viral vectors, adenoviral vectors,lentiviral vectors, and retroviral vectors.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs (or rAAV particles) of the disclosure arenot meant to be limiting and other suitable methods will be apparent tothe skilled artisan.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be “operably” linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., shRNA, miRNA, miRNA inhibitor).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present disclosuremay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. One possible intron sequence has the nucleotide sequence ofSEQ ID NO: 100. Another vector element that may be used is an internalribosome entry site (IRES). An IRES sequence is used to produce morethan one polypeptide from a single gene transcript. An IRES sequencewould be used to produce a protein that contains more than onepolypeptide chain. Selection of these and other common vector elementsis conventional, and many such sequences are available [see, e.g.,Sambrook et al. “Molecular Cloning. A Laboratory Manual”, 2d ed., ColdSpring Harbor Laboratory, New York (1989), and references cited thereinat, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, New York,1989]. In some embodiments, a Foot and Mouth Disease Virus 2A sequenceis included in polyprotein; this is a small peptide (approximately 18amino acids in length) that has been shown to mediate the cleavage ofpolyproteins (Ryan, M D et al., EMBO, 1994; 4: 928-933; Mattion, N M etal., J Virology, November 1996; p. 8124-8127; Furler, S et al., GeneTherapy, 2001; 8: 864-873; and Halpin, C et al., The Plant Journal,1999; 4: 453-459). The cleavage activity of the 2A sequence haspreviously been demonstrated in artificial systems including plasmidsand gene therapy vectors (AAV and retroviruses) (Ryan, M D et al., EMBO,1994; 4: 928-933; Mattion, N M et al., J Virology, November 1996; p.8124-8127; Furler, S et al., Gene Therapy, 2001; 8: 864-873; and Halpin,C et al., The Plant Journal, 1999; 4: 453-459; de Felipe, P et al., GeneTherapy, 1999; 6: 198-208; de Felipe, P et al., Human Gene Therapy,2000; 11: 1921-1931; and Klump, H et al., Gene Therapy, 2001; 8:811-817).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of thedisclosure may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al. (1985) Cell, 41:521-530], theSV40 promoter, the dihydrofolate reductase promoter, the .beta.-actinpromoter, the phosphoglycerol kinase (PGK) promoter (Gilham et al., J.Gene Med. 12(2):129-136, 2010), and the EF1.alpha. promoter[Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art.Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al. (1996) Proc. Natl. Acad. Sci. USA,93:3346-3351), the tetracycline-repressible system (Gossen et al. Proc.Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-induciblesystem (Gossen et al. Science, 268:1766-1769 (1995), see also Harvey etal. Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-induciblesystem (Wang et al. Nat. Biotech., 15:239-243 (1997) and Wang et al.Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magariet al. J. Clin. Invest., 100:2865-2872 (1997)). Still other types ofinducible promoters which may be useful in this context are those whichare regulated by a specific physiological state, e.g., temperature,acute phase, a particular differentiation state of the cell, or inreplicating cells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. Exemplary tissue-specific regulatory sequences include, butare not limited to the following tissue specific promoters: aliver-specific thyroxin binding globulin (TBG) promoter, an insulinpromoter, a glucagon promoter, a somatostatin promoter, a pancreaticpolypeptide (PPY) promoter, a synapsin-1 (Syn) promoter (Kugler et al.,Virology 311:89-95, 2003; Hioki et al., Gene Ther. 14:872-882, 2007;Kuroda et al., J. Gene Med 10:1163-1175, 2008), a creatine kinase (MCK)promoter (Wang et al., Gene Ther. 15:1489-1499, 2008; Talbot et al.,Mol. Ther. 18:601-608, 2010; Katwal et al., Gene Ther. 20(9):930-938,2013), a mammalian desmin (DES) promoter (Talbot et al., Mol. Ther.18:601-608, 2010), a C5-12 promoter (Wang et al., Gene Ther.15:1489-1499, 2008), an α-myosin heavy chain (a-MHC) promoter, a PDGFpromoter (Patterna, Gene Ther. 7(15):1304-1311, 2000; Hioki et al., GeneTher. 14:872-882, 2007), MecP2 promoter (Rastegar et al., PLoS One4:e6810, 2009; Gray et al., Human Gene Ther. 22:1143-1153, 2011), CaMKIIpromoter (Hioki et al., Gene Ther. 14:872-882, 2007; Kuroda et al., J.Gene Med 10:1163-1175, 2008), mGluR2 promoter (Brene et al., Eur. J.Eurosci. 12:1525-1533, 2000; Kuroda et al., J. Gene Med 10:1163-1175,2008), NFL promoter (Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xuet al., Gene Ther. 8:1323-1332, 2001), NFH promoter (Xu et al., HumanGene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332,2001), nJ2 promoter (Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xuet al., Gene Ther. 8:1323-1332, 2001), PPE promoter (Xu et al., HumanGene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332,2001), Enk promoter (Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xuet al., Gene Ther. 8:1323-1332, 2001), EAAT2 promoter (Su et al., Proc.Natl. Acad Sci. U.S.A. 100:1955-1960, 2003; Kuroda et al., J. Gene Med10:1163-1175, 2008), GFAP promoter (Brenner et al., J. Neurosci.14:1030-1037, 1994; Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xuet al., Gene Ther. 8:1323-1332, 2001; Lee et al., Glia 56:481-493, 2008;Dirren et al., Human Gene Ther. 25:109-120, 2014), MBP promoter (Chen etal., Gene Ther. 5(1):50-58, 1998), or a cardiac Troponin T (cTnT)promoter. Other exemplary promoters include Beta-actin promoter,hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9(1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. GeneTher., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol.Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al.,J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cellreceptor .alpha.-chain promoter, neuronal such as neuron-specificenolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol.,13:503-15 (1993); Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu etal., Gene Ther. 8:1323-1332, 2001), neurofilament light-chain genepromoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5(1991)), and the neuron-specific vgf gene promoter (Piccioli et al.,Neuron, 15:373-84 (1995)), among others which will be apparent to theskilled artisan.

In some embodiments, one or more bindings sites for one or more miRNAsare incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of an subjectharboring the transgene. The skilled artisan will appreciate thatbinding sites may be selected to control the expression of a transgenein a tissue specific manner. For example, binding sites for theliver-specific miR-122 may be incorporated into a transgene to inhibitexpression of that transgene in the liver. The target sites in the mRNAmay be in the 5′ UTR, the 3′ UTR or in the coding region. Typically, thetarget site is in the 3′ UTR of the mRNA. Furthermore, the transgene maybe designed such that multiple miRNAs regulate the mRNA by recognizingthe same or multiple sites. The presence of multiple miRNA binding sitesmay result in the cooperative action of multiple RISCs and providehighly efficient inhibition of expression. The target site sequence maycomprise a total of 5-100, 10-60, or more nucleotides. The target sitesequence may comprise at least 5 nucleotides of the sequence of a targetgene binding site.

Auditory Polypeptides and Auditory Polypeptide Transgene CodingSequences

The composition of the auditory polypeptide transgene sequence of therAAV vector will depend upon the use to which the resulting vector willbe generated. Thus, the disclosure embraces the delivery of rAAV vectorsencoding one or more auditory polypeptides, peptides, or proteins, whichare useful for the treatment or prevention of disease states associatedwith hearing loss in a mammalian subject. Exemplary therapeutic proteinsinclude one or more polypeptides selected from the group consisting ofotoferlin, Ca_(v)1.3, a scaffold protein selected from bassoon, piccolo,ribeye, and harmonin, Vglut3, synaptotagmin, a vesicle tethering/dockingprotein, a vesicle priming protein, a vesicle fusion protein, GluA2/3,or GluA4.

Optionally included in the AAV compositions are polypeptide reporterproteins. Reporter sequences that may be provided in a transgeneinclude, without limitation, DNA sequences encoding .beta.-lactamase,.beta.-galactosidase (LacZ), alkaline phosphatase, thymidine kinase,green fluorescent protein (GFP), chloramphenicol acetyltransferase(CAT), luciferase, and others well known in the art. When associatedwith regulatory elements which drive their expression, the reportersequences provide signals detectable by conventional means, includingenzymatic, radiographic, colorimetric, fluorescence or otherspectrographic assays, fluorescent activating cell sorting assays, andimmunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for J-galactosidase activity.Where the transgene is green fluorescent protein or luciferase, thevector carrying the signal may be measured visually by color or lightproduction in a luminometer. Such reporters can, for example, be usefulin verifying the tissue-specific targeting capabilities and tissuespecific promoter regulatory activity of an rAAV.

In another example, the transgene encodes a protein or functional RNAthat is intended to be used to create an animal model of disease.Appropriate transgene coding sequences will be apparent to the skilledartisan.

The rAAV vectors may comprise a gene or a portion of a gene encoding anauditory polypeptide, to be transferred to a subject to treat a diseaseassociated with reduced expression, lack of expression or dysfunction ofthe auditory polypeptide gene or another gene in the functional pathwayof an auditory polypeptide.

In a first aspect, provided are therapeutic compositions including aplurality of adeno-associated viral (AAV) vectors, wherein the pluralityof AAV vectors are capable of constituting an auditory polypeptidemessenger RNA in a target cell of a human subject to whom thetherapeutic composition is administered. Preferably, the plurality ofAAV vectors are capable of constituting a full-length auditorypolypeptide messenger RNA in a target cell of a human subject to whomthe therapeutic composition is administered. The plurality of AAVvectors include a first AAV vector and a second AAV vector, wherein thefirst and second AAV vectors independently contain packaging capacity ofless than about 6 kb. The auditory polypeptide messenger RNA encodes anauditory polypeptide selected from the group consisting of otoferlin andan ortholog or homolog thereof, for example, as provided herein. The AAVvectors contain at least one promoter sequence selected from a CBA, aCMV, or a CB7 promoter, or one or more Cochlea-specific promoters. Insome embodiments, an AAV vector can include a CMV enhancer and promotersequence, e.g., SEQ ID NO: 70. In some embodiments, an AAV vector caninclude a CMV enhancer and a chicken β-actin (CBA) promoter, e.g., SEQID NO: 61. In some embodiments, an AAV vector can include a CMVdpromoter sequence, e.g., SEQ ID NO: 86. In some embodiments, an AAVvector can include a promoter that comprises a CMV enhancer and a CBApromoter, e.g., the nucleotide sequences of SEQ ID NO: 98 and 99. Insome embodiments, the nucleotide sequence of SEQ ID NO: 98 precedes thenucleotide sequence of SEQ ID NO: 99 and is optionally followed by achimeric intron, e.g., the nucleotide sequence of SEQ ID NO: 100.

The compositions provided herein include at least two (e.g., two, three,four, five, or six) nucleic acid vectors, where: each of the at leasttwo different vectors includes a coding sequence that encodes adifferent portion of an otoferlin protein, each of the encoded portionsbeing at least 30 amino acids (e.g., between about 30 amino acids toabout 1950 amino acids, about 30 amino acids to about 1900 amino acids,about 30 amino acids to about 1850 amino acids, about 30 amino acids toabout 1800 amino acids, about 30 amino acids to about 1750 amino acids,about 30 amino acids to about 1700 amino acids, about 30 amino acids toabout 1650 amino acids, about 30 amino acids to about 1600 amino acids,about 30 amino acids to about 1550 amino acids, about 30 amino acids toabout 1500 amino acids, about 30 amino acids to about 1450 amino acids,about 30 amino acids to about 1400 amino acids, about 30 amino acids toabout 1350 amino acids, about 30 amino acids to about 1300 amino acids,about 30 amino acids to about 1250 amino acids, about 30 amino acids toabout 1200 amino acids, about 30 amino acids to about 1150 amino acids,about 30 amino acids to about 1100 amino acids, about 30 amino acids toabout 1050 amino acids, about 30 amino acids to about 1000 amino acids,about 30 amino acids to about 950 amino acids, about 30 amino acids toabout 900 amino acids, about 30 amino acids to about 850 amino acids,about 30 amino acids to about 800 amino acids, about 30 amino acids toabout 750 amino acids, about 30 amino acids to about 700 amino acids,about 30 amino acids to about 650 amino acids, about 30 amino acids toabout 600 amino acids, about 30 amino acids to about 550 amino acids,about 30 amino acids to about 500 amino acids, about 30 amino acids toabout 450 amino acids, about 30 amino acids to about 400 amino acids,about 30 amino acids to about 350 amino acids, about 30 amino acids toabout 300 amino acids, about 30 amino acids to about 250 amino acids,about 30 amino acids to about 200 amino acids, about 30 amino acids toabout 150 amino acids, about 30 amino acids to about 100 amino acids,about 30 amino acids to about 50 amino acids, about 50 amino acids toabout 1950 amino acids, about 50 amino acids to about 1900 amino acids,about 50 amino acids to about 1850 amino acids, about 50 amino acids toabout 1800 amino acids, about 50 amino acids to about 1750 amino acids,about 50 amino acids to about 1700 amino acids, about 50 amino acids toabout 1650 amino acids, about 50 amino acids to about 1600 amino acids,about 50 amino acids to about 1550 amino acids, about 50 amino acids toabout 1500 amino acids, about 50 amino acids to about 1450 amino acids,about 50 amino acids to about 1400 amino acids, about 50 amino acids toabout 1350 amino acids, about 50 amino acids to about 1300 amino acids,about 50 amino acids to about 1250 amino acids, about 50 amino acids toabout 1200 amino acids, about 50 amino acids to about 1150 amino acids,about 50 amino acids to about 1100 amino acids, about 50 amino acids toabout 1050 amino acids, about 50 amino acids to about 1000 amino acids,about 50 amino acids to about 950 amino acids, about 50 amino acids toabout 900 amino acids, about 50 amino acids to about 850 amino acids,about 50 amino acids to about 800 amino acids, about 50 amino acids toabout 750 amino acids, about 50 amino acids to about 700 amino acids,about 50 amino acids to about 650 amino acids, about 50 amino acids toabout 600 amino acids, about 50 amino acids to about 550 amino acids,about 50 amino acids to about 500 amino acids, about 50 amino acids toabout 450 amino acids, about 50 amino acids to about 400 amino acids,about 50 amino acids to about 350 amino acids, about 50 amino acids toabout 300 amino acids, about 50 amino acids to about 250 amino acids,about 50 amino acids to about 200 amino acids, about 50 amino acids toabout 150 amino acids, about 50 amino acids to about 100 amino acids,about 100 amino acids to about 1950 amino acids, about 100 amino acidsto about 1900 amino acids, about 100 amino acids to about 1850 aminoacids, about 100 amino acids to about 1800 amino acids, about 100 aminoacids to about 1750 amino acids, about 100 amino acids to about 1700amino acids, about 100 amino acids to about 1650 amino acids, about 100amino acids to about 1600 amino acids, about 100 amino acids to about1550 amino acids, about 100 amino acids to about 1500 amino acids, about100 amino acids to about 1450 amino acids, about 100 amino acids toabout 1400 amino acids, about 100 amino acids to about 1350 amino acids,about 100 amino acids to about 1300 amino acids, about 100 amino acidsto about 1250 amino acids, about 100 amino acids to about 1200 aminoacids, about 100 amino acids to about 1150 amino acids, about 100 aminoacids to about 1100 amino acids, about 100 amino acids to about 1050amino acids, about 100 amino acids to about 1000 amino acids, about 100amino acids to about 950 amino acids, about 100 amino acids to about 900amino acids, about 100 amino acids to about 850 amino acids, about 100amino acids to about 800 amino acids, about 100 amino acids to about 750amino acids, about 100 amino acids to about 700 amino acids, about 100amino acids to about 650 amino acids, about 100 amino acids to about 600amino acids, about 100 amino acids to about 550 amino acids, about 100amino acids to about 500 amino acids, about 100 amino acids to about 450amino acids, about 100 amino acids to about 400 amino acids, about 100amino acids to about 350 amino acids, about 100 amino acids to about 300amino acids, about 100 amino acids to about 250 amino acids, about 100amino acids to about 200 amino acids, about 100 amino acids to about 150amino acids, about 150 amino acids to about 1950 amino acids, about 150amino acids to about 1900 amino acids, about 150 amino acids to about1850 amino acids, about 150 amino acids to about 1800 amino acids, about150 amino acids to about 1750 amino acids, about 150 amino acids toabout 1700 amino acids, about 150 amino acids to about 1650 amino acids,about 150 amino acids to about 1600 amino acids, about 150 amino acidsto about 1550 amino acids, about 150 amino acids to about 1500 aminoacids, about 150 amino acids to about 1450 amino acids, about 150 aminoacids to about 1400 amino acids, about 150 amino acids to about 1350amino acids, about 150 amino acids to about 1300 amino acids, about 150amino acids to about 1250 amino acids, about 150 amino acids to about1200 amino acids, about 150 amino acids to about 1150 amino acids, about150 amino acids to about 1100 amino acids, about 150 amino acids toabout 1050 amino acids, about 150 amino acids to about 1000 amino acids,about 150 amino acids to about 950 amino acids, about 150 amino acids toabout 900 amino acids, about 150 amino acids to about 850 amino acids,about 150 amino acids to about 800 amino acids, about 150 amino acids toabout 750 amino acids, about 150 amino acids to about 700 amino acids,about 150 amino acids to about 650 amino acids, about 150 amino acids toabout 600 amino acids, about 150 amino acids to about 550 amino acids,about 150 amino acids to about 500 amino acids, about 150 amino acids toabout 450 amino acids, about 150 amino acids to about 400 amino acids,about 150 amino acids to about 350 amino acids, about 150 amino acids toabout 300 amino acids, about 150 amino acids to about 250 amino acids,about 150 amino acids to about 200 amino acids, about 200 amino acids toabout 1950 amino acids, about 200 amino acids to about 1900 amino acids,about 200 amino acids to about 1850 amino acids, about 200 amino acidsto about 1800 amino acids, about 200 amino acids to about 1750 aminoacids, about 200 amino acids to about 1700 amino acids, about 200 aminoacids to about 1650 amino acids, about 200 amino acids to about 1600amino acids, about 200 amino acids to about 1550 amino acids, about 200amino acids to about 1500 amino acids, about 200 amino acids to about1450 amino acids, about 200 amino acids to about 1400 amino acids, about200 amino acids to about 1350 amino acids, about 200 amino acids toabout 1300 amino acids, about 200 amino acids to about 1250 amino acids,about 200 amino acids to about 1200 amino acids, about 200 amino acidsto about 1150 amino acids, about 200 amino acids to about 1100 aminoacids, about 200 amino acids to about 1050 amino acids, about 200 aminoacids to about 1000 amino acids, about 200 amino acids to about 950amino acids, about 200 amino acids to about 900 amino acids, about 200amino acids to about 850 amino acids, about 200 amino acids to about 800amino acids, about 200 amino acids to about 750 amino acids, about 200amino acids to about 700 amino acids, about 200 amino acids to about 650amino acids, about 200 amino acids to about 600 amino acids, about 200amino acids to about 550 amino acids, about 200 amino acids to about 500amino acids, about 200 amino acids to about 450 amino acids, about 200amino acids to about 400 amino acids, about 200 amino acids to about 350amino acids, about 200 amino acids to about 300 amino acids, about 200amino acids to about 250 amino acids, about 250 amino acids to about1950 amino acids, about 250 amino acids to about 1900 amino acids, about250 amino acids to about 1850 amino acids, about 250 amino acids toabout 1800 amino acids, about 250 amino acids to about 1750 amino acids,about 250 amino acids to about 1700 amino acids, about 250 amino acidsto about 1650 amino acids, about 250 amino acids to about 1600 aminoacids, about 250 amino acids to about 1550 amino acids, about 250 aminoacids to about 1500 amino acids, about 250 amino acids to about 1450amino acids, about 250 amino acids to about 1400 amino acids, about 250amino acids to about 1350 amino acids, about 250 amino acids to about1300 amino acids, about 250 amino acids to about 1250 amino acids, about250 amino acids to about 1200 amino acids, about 250 amino acids toabout 1150 amino acids, about 250 amino acids to about 1100 amino acids,about 250 amino acids to about 1050 amino acids, about 250 amino acidsto about 1000 amino acids, about 250 amino acids to about 950 aminoacids, about 250 amino acids to about 900 amino acids, about 250 aminoacids to about 850 amino acids, about 250 amino acids to about 800 aminoacids, about 250 amino acids to about 750 amino acids, about 250 aminoacids to about 700 amino acids, about 250 amino acids to about 650 aminoacids, about 250 amino acids to about 600 amino acids, about 250 aminoacids to about 550 amino acids, about 250 amino acids to about 500 aminoacids, about 250 amino acids to about 450 amino acids, about 250 aminoacids to about 400 amino acids, about 250 amino acids to about 350 aminoacids, about 250 amino acids to about 300 amino acids, about 300 aminoacids to about 1950 amino acids, about 300 amino acids to about 1900amino acids, about 300 amino acids to about 1850 amino acids, about 300amino acids to about 1800 amino acids, about 300 amino acids to about1750 amino acids, about 300 amino acids to about 1700 amino acids, about300 amino acids to about 1650 amino acids, about 300 amino acids toabout 1600 amino acids, about 300 amino acids to about 1550 amino acids,about 300 amino acids to about 1500 amino acids, about 300 amino acidsto about 1450 amino acids, about 300 amino acids to about 1400 aminoacids, about 300 amino acids to about 1350 amino acids, about 300 aminoacids to about 1300 amino acids, about 300 amino acids to about 1250amino acids, about 300 amino acids to about 1200 amino acids, about 300amino acids to about 1150 amino acids, about 300 amino acids to about1100 amino acids, about 300 amino acids to about 1050 amino acids, about300 amino acids to about 1000 amino acids, about 300 amino acids toabout 950 amino acids, about 300 amino acids to about 900 amino acids,about 300 amino acids to about 850 amino acids, about 300 amino acids toabout 800 amino acids, about 300 amino acids to about 750 amino acids,about 300 amino acids to about 700 amino acids, about 300 amino acids toabout 650 amino acids, about 300 amino acids to about 600 amino acids,about 300 amino acids to about 550 amino acids, about 300 amino acids toabout 500 amino acids, about 300 amino acids to about 450 amino acids,about 300 amino acids to about 400 amino acids, about 300 amino acids toabout 350 amino acids, about 350 amino acids to about 1950 amino acids,about 350 amino acids to about 1900 amino acids, about 350 amino acidsto about 1850 amino acids, about 350 amino acids to about 1800 aminoacids, about 350 amino acids to about 1750 amino acids, about 350 aminoacids to about 1700 amino acids, about 350 amino acids to about 1650amino acids, about 350 amino acids to about 1600 amino acids, about 350amino acids to about 1550 amino acids, about 350 amino acids to about1500 amino acids, about 350 amino acids to about 1450 amino acids, about350 amino acids to about 1400 amino acids, about 350 amino acids toabout 1350 amino acids, about 350 amino acids to about 1300 amino acids,about 350 amino acids to about 1250 amino acids, about 350 amino acidsto about 1200 amino acids, about 350 amino acids to about 1150 aminoacids, about 350 amino acids to about 1100 amino acids, about 350 aminoacids to about 1050 amino acids, about 350 amino acids to about 1000amino acids, about 350 amino acids to about 950 amino acids, about 350amino acids to about 900 amino acids, about 350 amino acids to about 850amino acids, about 350 amino acids to about 800 amino acids, about 350amino acids to about 750 amino acids, about 350 amino acids to about 700amino acids, about 350 amino acids to about 650 amino acids, about 350amino acids to about 600 amino acids, about 350 amino acids to about 550amino acids, about 350 amino acids to about 500 amino acids, about 350amino acids to about 450 amino acids, about 350 amino acids to about 400amino acids, about 400 amino acids to about 1950 amino acids, about 400amino acids to about 1900 amino acids, about 400 amino acids to about1850 amino acids, about 400 amino acids to about 1800 amino acids, about400 amino acids to about 1750 amino acids, about 400 amino acids toabout 1700 amino acids, about 400 amino acids to about 1650 amino acids,about 400 amino acids to about 1600 amino acids, about 400 amino acidsto about 1550 amino acids, about 400 amino acids to about 1500 aminoacids, about 400 amino acids to about 1450 amino acids, about 400 aminoacids to about 1400 amino acids, about 400 amino acids to about 1350amino acids, about 400 amino acids to about 1300 amino acids, about 400amino acids to about 1250 amino acids, about 400 amino acids to about1200 amino acids, about 400 amino acids to about 1150 amino acids, about400 amino acids to about 1100 amino acids, about 400 amino acids toabout 1050 amino acids, about 400 amino acids to about 1000 amino acids,about 400 amino acids to about 950 amino acids, about 400 amino acids toabout 900 amino acids, about 400 amino acids to about 850 amino acids,about 400 amino acids to about 800 amino acids, about 400 amino acids toabout 750 amino acids, about 400 amino acids to about 700 amino acids,about 400 amino acids to about 650 amino acids, about 400 amino acids toabout 600 amino acids, about 400 amino acids to about 550 amino acids,about 400 amino acids to about 500 amino acids, about 400 amino acids toabout 450 amino acids, about 450 amino acids to about 1950 amino acids,about 450 amino acids to about 1900 amino acids, about 450 amino acidsto about 1850 amino acids, about 450 amino acids to about 1800 aminoacids, about 450 amino acids to about 1750 amino acids, about 450 aminoacids to about 1700 amino acids, about 450 amino acids to about 1650amino acids, about 450 amino acids to about 1600 amino acids, about 450amino acids to about 1550 amino acids, about 450 amino acids to about1500 amino acids, about 450 amino acids to about 1450 amino acids, about450 amino acids to about 1400 amino acids, about 450 amino acids toabout 1350 amino acids, about 450 amino acids to about 1300 amino acids,about 450 amino acids to about 1250 amino acids, about 450 amino acidsto about 1200 amino acids, about 450 amino acids to about 1150 aminoacids, about 450 amino acids to about 1100 amino acids, about 450 aminoacids to about 1050 amino acids, about 450 amino acids to about 1000amino acids, about 450 amino acids to about 950 amino acids, about 450amino acids to about 900 amino acids, about 450 amino acids to about 850amino acids, about 450 amino acids to about 800 amino acids, about 450amino acids to about 750 amino acids, about 450 amino acids to about 700amino acids, about 450 amino acids to about 650 amino acids, about 450amino acids to about 600 amino acids, about 450 amino acids to about 550amino acids, about 450 amino acids to about 500 amino acids, about 500amino acids to about 1950 amino acids, about 500 amino acids to about1900 amino acids, about 500 amino acids to about 1850 amino acids, about500 amino acids to about 1800 amino acids, about 500 amino acids toabout 1750 amino acids, about 500 amino acids to about 1700 amino acids,about 500 amino acids to about 1650 amino acids, about 500 amino acidsto about 1600 amino acids, about 500 amino acids to about 1550 aminoacids, about 500 amino acids to about 1500 amino acids, about 500 aminoacids to about 1450 amino acids, about 500 amino acids to about 1400amino acids, about 500 amino acids to about 1350 amino acids, about 500amino acids to about 1300 amino acids, about 500 amino acids to about1250 amino acids, about 500 amino acids to about 1200 amino acids, about500 amino acids to about 1150 amino acids, about 500 amino acids toabout 1100 amino acids, about 500 amino acids to about 1050 amino acids,about 500 amino acids to about 1000 amino acids, about 500 amino acidsto about 950 amino acids, about 500 amino acids to about 900 aminoacids, about 500 amino acids to about 850 amino acids, about 500 aminoacids to about 800 amino acids, about 500 amino acids to about 750 aminoacids, about 500 amino acids to about 700 amino acids, about 500 aminoacids to about 650 amino acids, about 500 amino acids to about 600 aminoacids, about 500 amino acids to about 550 amino acids, about 550 aminoacids to about 1950 amino acids, about 550 amino acids to about 1900amino acids, about 550 amino acids to about 1850 amino acids, about 550amino acids to about 1800 amino acids, about 550 amino acids to about1750 amino acids, about 550 amino acids to about 1700 amino acids, about550 amino acids to about 1650 amino acids, about 550 amino acids toabout 1600 amino acids, about 550 amino acids to about 1550 amino acids,about 550 amino acids to about 1500 amino acids, about 550 amino acidsto about 1450 amino acids, about 550 amino acids to about 1400 aminoacids, about 550 amino acids to about 1350 amino acids, about 550 aminoacids to about 1300 amino acids, about 550 amino acids to about 1250amino acids, about 550 amino acids to about 1200 amino acids, about 550amino acids to about 1150 amino acids, about 550 amino acids to about1100 amino acids, about 550 amino acids to about 1050 amino acids, about550 amino acids to about 1000 amino acids, about 550 amino acids toabout 950 amino acids, about 550 amino acids to about 900 amino acids,about 550 amino acids to about 850 amino acids, about 550 amino acids toabout 800 amino acids, about 550 amino acids to about 750 amino acids,about 550 amino acids to about 700 amino acids, about 550 amino acids toabout 650 amino acids, about 550 amino acids to about 600 amino acids,about 600 amino acids to about 1950 amino acids, about 600 amino acidsto about 1900 amino acids, about 600 amino acids to about 1850 aminoacids, about 600 amino acids to about 1800 amino acids, about 600 aminoacids to about 1750 amino acids, about 600 amino acids to about 1700amino acids, about 600 amino acids to about 1650 amino acids, about 600amino acids to about 1600 amino acids, about 600 amino acids to about1550 amino acids, about 600 amino acids to about 1500 amino acids, about600 amino acids to about 1450 amino acids, about 600 amino acids toabout 1400 amino acids, about 600 amino acids to about 1350 amino acids,about 600 amino acids to about 1300 amino acids, about 600 amino acidsto about 1250 amino acids, about 600 amino acids to about 1200 aminoacids, about 600 amino acids to about 1150 amino acids, about 600 aminoacids to about 1100 amino acids, about 600 amino acids to about 1050amino acids, about 600 amino acids to about 1000 amino acids, about 600amino acids to about 950 amino acids, about 600 amino acids to about 900amino acids, about 600 amino acids to about 850 amino acids, about 600amino acids to about 800 amino acids, about 600 amino acids to about 750amino acids, about 600 amino acids to about 700 amino acids, about 600amino acids to about 650 amino acids, about 650 amino acids to about1950 amino acids, about 650 amino acids to about 1900 amino acids, about650 amino acids to about 1850 amino acids, about 650 amino acids toabout 1800 amino acids, about 650 amino acids to about 1750 amino acids,about 650 amino acids to about 1700 amino acids, about 650 amino acidsto about 1650 amino acids, about 650 amino acids to about 1600 aminoacids, about 650 amino acids to about 1550 amino acids, about 650 aminoacids to about 1500 amino acids, about 650 amino acids to about 1450amino acids, about 650 amino acids to about 1400 amino acids, about 650amino acids to about 1350 amino acids, about 650 amino acids to about1300 amino acids, about 650 amino acids to about 1250 amino acids, about650 amino acids to about 1200 amino acids, about 650 amino acids toabout 1150 amino acids, about 650 amino acids to about 1100 amino acids,about 650 amino acids to about 1050 amino acids, about 650 amino acidsto about 1000 amino acids, about 650 amino acids to about 950 aminoacids, about 650 amino acids to about 900 amino acids, about 650 aminoacids to about 850 amino acids, about 650 amino acids to about 800 aminoacids, about 650 amino acids to about 750 amino acids, about 650 aminoacids to about 700 amino acids, about 700 amino acids to about 1950amino acids, about 700 amino acids to about 1900 amino acids, about 700amino acids to about 1850 amino acids, about 700 amino acids to about1800 amino acids, about 700 amino acids to about 1750 amino acids, about700 amino acids to about 1700 amino acids, about 700 amino acids toabout 1650 amino acids, about 700 amino acids to about 1600 amino acids,about 700 amino acids to about 1550 amino acids, about 700 amino acidsto about 1500 amino acids, about 700 amino acids to about 1450 aminoacids, about 700 amino acids to about 1400 amino acids, about 700 aminoacids to about 1350 amino acids, about 700 amino acids to about 1300amino acids, about 700 amino acids to about 1250 amino acids, about 700amino acids to about 1200 amino acids, about 700 amino acids to about1150 amino acids, about 700 amino acids to about 1100 amino acids, about700 amino acids to about 1050 amino acids, about 700 amino acids toabout 1000 amino acids, about 700 amino acids to about 950 amino acids,about 700 amino acids to about 900 amino acids, about 700 amino acids toabout 850 amino acids, about 700 amino acids to about 800 amino acids,about 700 amino acids to about 750 amino acids, about 750 amino acids toabout 1950 amino acids, about 750 amino acids to about 1900 amino acids,about 750 amino acids to about 1850 amino acids, about 750 amino acidsto about 1800 amino acids, about 750 amino acids to about 1750 aminoacids, about 750 amino acids to about 1700 amino acids, about 750 aminoacids to about 1650 amino acids, about 750 amino acids to about 1600amino acids, about 750 amino acids to about 1550 amino acids, about 750amino acids to about 1500 amino acids, about 750 amino acids to about1450 amino acids, about 750 amino acids to about 1400 amino acids, about750 amino acids to about 1350 amino acids, about 750 amino acids toabout 1250 amino acids, about 750 amino acids to about 1200 amino acids,about 750 amino acids to about 1150 amino acids, about 750 amino acidsto about 1100 amino acids, about 750 amino acids to about 1050 aminoacids, about 750 amino acids to about 1000 amino acids, about 750 aminoacids to about 950 amino acids, about 750 amino acids to about 900 aminoacids, about 750 amino acids to about 850 amino acids, about 750 aminoacids to about 800 amino acids, about 800 amino acids to about 1950amino acids, about 800 amino acids to about 1900 amino acids, about 800amino acids to about 1850 amino acids, about 800 amino acids to about1800 amino acids, about 800 amino acids to about 1750 amino acids, about800 amino acids to about 1700 amino acids, about 800 amino acids toabout 1650 amino acids, about 800 amino acids to about 1600 amino acids,about 800 amino acids to about 1550 amino acids, about 800 amino acidsto about 1500 amino acids, about 800 amino acids to about 1450 aminoacids, about 800 amino acids to about 1400 amino acids, about 800 aminoacids to about 1350 amino acids, about 800 amino acids to about 1300amino acids, about 800 amino acids to about 1250 amino acids, about 800amino acids to about 1200 amino acids, about 800 amino acids to about1150 amino acids, about 800 amino acids to about 1100 amino acids, about800 amino acids to about 1050 amino acids, about 800 amino acids toabout 1000 amino acids, about 800 amino acids to about 950 amino acids,about 800 amino acids to about 900 amino acids, about 800 amino acids toabout 850 amino acids, about 850 amino acids to about 1950 amino acids,about 850 amino acids to about 1900 amino acids, about 850 amino acidsto about 1850 amino acids, about 850 amino acids to about 1800 aminoacids, about 850 amino acids to about 1750 amino acids, about 850 aminoacids to about 1700 amino acids, about 850 amino acids to about 1650amino acids, about 850 amino acids to about 1600 amino acids, about 850amino acids to about 1550 amino acids, about 850 amino acids to about1500 amino acids, about 850 amino acids to about 1450 amino acids, about850 amino acids to about 1400 amino acids, about 850 amino acids toabout 1350 amino acids, about 850 amino acids to about 1300 amino acids,about 850 amino acids to about 1250 amino acids, about 850 amino acidsto about 1200 amino acids, about 850 amino acids to about 1150 aminoacids, about 850 amino acids to about 1100 amino acids, about 850 aminoacids to about 1050 amino acids, about 850 amino acids to about 1000amino acids, about 850 amino acids to about 950 amino acids, about 850amino acids to about 900 amino acids, about 900 amino acids to about1950 amino acids, about 900 amino acids to about 1900 amino acids, about900 amino acids to about 1850 amino acids, about 900 amino acids toabout 1800 amino acids, about 900 amino acids to about 1750 amino acids,about 900 amino acids to about 1700 amino acids, about 900 amino acidsto about 1650 amino acids, about 900 amino acids to about 1600 aminoacids, about 900 amino acids to about 1550 amino acids, about 900 aminoacids to about 1500 amino acids, about 900 amino acids to about 1450amino acids, about 900 amino acids to about 1400 amino acids, about 900amino acids to about 1350 amino acids, about 900 amino acids to about1300 amino acids, about 900 amino acids to about 1250 amino acids, about900 amino acids to about 1200 amino acids, about 900 amino acids toabout 1150 amino acids, about 900 amino acids to about 1100 amino acids,about 900 amino acids to about 1050 amino acids, about 900 amino acidsto about 1000 amino acids, about 900 amino acids to about 950 aminoacids, about 950 amino acids to about 1950 amino acids, about 950 aminoacids to about 1900 amino acids, about 950 amino acids to about 1850amino acids, about 950 amino acids to about 1800 amino acids, about 950amino acids to about 1750 amino acids, about 950 amino acids to about1700 amino acids, about 950 amino acids to about 1650 amino acids, about950 amino acids to about 1600 amino acids, about 950 amino acids toabout 1550 amino acids, about 950 amino acids to about 1500 amino acids,about 950 amino acids to about 1450 amino acids, about 950 amino acidsto about 1400 amino acids, about 950 amino acids to about 1350 aminoacids, about 950 amino acids to about 1300 amino acids, about 950 aminoacids to about 1250 amino acids, about 950 amino acids to about 1200amino acids, about 950 amino acids to about 1150 amino acids, about 950amino acids to about 1100 amino acids, about 950 amino acids to about1050 amino acids, about 950 amino acids to about 1000 amino acids, about1000 amino acids to about 1950 amino acids, about 1000 amino acids toabout 1900 amino acids, about 1000 amino acids to about 1850 aminoacids, about 1000 amino acids to about 1800 amino acids, about 1000amino acids to about 1750 amino acids, about 1000 amino acids to about1700 amino acids, about 1000 amino acids to about 1650 amino acids,about 1000 amino acids to about 1600 amino acids, about 1000 amino acidsto about 1550 amino acids, about 1000 amino acids to about 1500 aminoacids, about 1000 amino acids to about 1450 amino acids, about 1000amino acids to about 1400 amino acids, about 1000 amino acids to about1350 amino acids, about 1000 amino acids to about 1300 amino acids,about 1000 amino acids to about 1250 amino acids, about 1000 amino acidsto about 1200 amino acids, about 1000 amino acids to about 1150 aminoacids, about 1000 amino acids to about 1100 amino acids, about 1000amino acids to about 1050 amino acids, about 1050 amino acids to about1950 amino acids, about 1050 amino acids to about 1900 amino acids,about 1050 amino acids to about 1850 amino acids, about 1050 amino acidsto about 1800 amino acids, about 1050 amino acids to about 1750 aminoacids, about 1050 amino acids to about 1700 amino acids, about 1050amino acids to about 1650 amino acids, about 1050 amino acids to about1600 amino acids, about 1050 amino acids to about 1550 amino acids,about 1050 amino acids to about 1500 amino acids, about 1050 amino acidsto about 1450 amino acids, about 1050 amino acids to about 1400 aminoacids, about 1050 amino acids to about 1350 amino acids, about 1050amino acids to about 1300 amino acids, about 1050 amino acids to about1250 amino acids, about 1050 amino acids to about 1200 amino acids,about 1050 amino acids to about 1150 amino acids, about 1050 amino acidsto about 1100 amino acids, about 1100 amino acids to about 1950 aminoacids, about 1100 amino acids to about 1900 amino acids, about 1100amino acids to about 1850 amino acids, about 1100 amino acids to about1800 amino acids, about 1100 amino acids to about 1750 amino acids,about 1100 amino acids to about 1700 amino acids, about 1100 amino acidsto about 1650 amino acids, about 1100 amino acids to about 1600 aminoacids, about 1100 amino acids to about 1550 amino acids, about 1100amino acids to about 1500 amino acids, about 1100 amino acids to about1450 amino acids, about 1100 amino acids to about 1400 amino acids,about 1100 amino acids to about 1350 amino acids, about 1100 amino acidsto about 1300 amino acids, about 1100 amino acids to about 1250 aminoacids, about 1100 amino acids to about 1200 amino acids, about 1100amino acids to about 1150 amino acids, about 1150 amino acids to about1950 amino acids, about 1150 amino acids to about 1900 amino acids,about 1150 amino acids to about 1850 amino acids, about 1150 amino acidsto about 1800 amino acids, about 1150 amino acids to about 1750 aminoacids, about 1150 amino acids to about 1700 amino acids, about 1150amino acids to about 1650 amino acids, about 1150 amino acids to about1600 amino acids, about 1150 amino acids to about 1550 amino acids,about 1150 amino acids to about 1500 amino acids, about 1150 amino acidsto about 1450 amino acids, about 1150 amino acids to about 1400 aminoacids, about 1150 amino acids to about 1350 amino acids, about 1150amino acids to about 1300 amino acids, about 1150 amino acids to about1250 amino acids, about 1150 amino acids to about 1200 amino acids,about 1200 amino acids to about 1950 amino acids, about 1200 amino acidsto about 1900 amino acids, about 1200 amino acids to about 1850 aminoacids, about 1200 amino acids to about 1800 amino acids, about 1200amino acids to about 1750 amino acids, about 1200 amino acids to about1700 amino acids, about 1200 amino acids to about 1650 amino acids,about 1200 amino acids to about 1600 amino acids, about 1200 amino acidsto about 1550 amino acids, about 1200 amino acids to about 1500 aminoacids, about 1200 amino acids to about 1450 amino acids, about 1200amino acids to about 1400 amino acids, about 1200 amino acids to about1350 amino acids, about 1200 amino acids to about 1300 amino acids,about 1200 amino acids to about 1250 amino acids, about 1250 amino acidsto about 1950 amino acids, about 1250 amino acids to about 1900 aminoacids, about 1250 amino acids to about 1850 amino acids, about 1250amino acids to about 1800 amino acids, about 1250 amino acids to about1750 amino acids, about 1250 amino acids to about 1700 amino acids,about 1250 amino acids to about 1650 amino acids, about 1250 amino acidsto about 1600 amino acids, about 1250 amino acids to about 1550 aminoacids, about 1250 amino acids to about 1500 amino acids, about 1250amino acids to about 1450 amino acids, about 1250 amino acids to about1400 amino acids, about 1250 amino acids to about 1350 amino acids,about 1250 amino acids to about 1300 amino acids, about 1300 amino acidsto about 1950 amino acids, about 1300 amino acids to about 1900 aminoacids, about 1300 amino acids to about 1850 amino acids, about 1300amino acids to about 1800 amino acids, about 1300 amino acids to about1750 amino acids, about 1300 amino acids to about 1700 amino acids,about 1300 amino acids to about 1650 amino acids, about 1300 amino acidsto about 1600 amino acids, about 1300 amino acids to about 1550 aminoacids, about 1300 amino acids to about 1500 amino acids, about 1300amino acids to about 1450 amino acids, about 1300 amino acids to about1400 amino acids, about 1300 amino acids to about 1350 amino acids,about 1350 amino acids to about 1950 amino acids, about 1350 amino acidsto about 1900 amino acids, about 1350 amino acids to about 1850 aminoacids, about 1350 amino acids to about 1800 amino acids, about 1350amino acids to about 1750 amino acids, about 1350 amino acids to about1700 amino acids, about 1350 amino acids to about 1650 amino acids,about 1350 amino acids to about 1600 amino acids, about 1350 amino acidsto about 1550 amino acids, about 1350 amino acids to about 1500 aminoacids, about 1350 amino acids to about 1450 amino acids, about 1350amino acids to about 1400 amino acids, about 1400 amino acids to about1950 amino acids, about 1400 amino acids to about 1900 amino acids,about 1400 amino acids to about 1850 amino acids, about 1400 amino acidsto about 1800 amino acids, about 1400 amino acids to about 1750 aminoacids, about 1400 amino acids to about 1700 amino acids, about 1400amino acids to about 1650 amino acids, about 1400 amino acids to about1600 amino acids, about 1400 amino acids to about 1550 amino acids,about 1400 amino acids to about 1500 amino acids, about 1400 amino acidsto about 1450 amino acids, about 1450 amino acids to about 1950 aminoacids, about 1450 amino acids to about 1900 amino acids, about 1450amino acids to about 1850 amino acids, about 1450 amino acids to about1800 amino acids, about 1450 amino acids to about 1750 amino acids,about 1450 amino acids to about 1700 amino acids, about 1450 amino acidsto about 1650 amino acids, about 1450 amino acids to about 1600 aminoacids, about 1450 amino acids to about 1550 amino acids, about 1450amino acids to about 1500 amino acids, about 1600 amino acids to about1950 amino acids, about 1600 amino acids to about 1900 amino acids,about 1600 amino acids to about 1850 amino acids, about 1600 amino acidsto about 1800 amino acids, about 1600 amino acids to about 1750 aminoacids, about 1600 amino acids to about 1700 amino acids, about 1600amino acids to about 1650 amino acids, about 1500 amino acids to about1950 amino acids, about 1500 amino acids to about 1900 amino acids,about 1500 amino acids to about 1850 amino acids, about 1500 amino acidsto about 1800 amino acids, about 1500 amino acids to about 1750 aminoacids, about 1500 amino acids to about 1700 amino acids, about 1500amino acids to about 1650 amino acids, about 1500 amino acids to about1600 amino acids, about 1500 amino acids to about 1550 amino acids,about 1550 amino acids to about 1950 amino acids, about 1550 amino acidsto about 1900 amino acids, about 1550 amino acids to about 1850 aminoacids, about 1550 amino acids to about 1800 amino acids, about 1550amino acids to about 1750 amino acids, about 1550 amino acids to about1700 amino acids, about 1550 amino acids to about 1650 amino acids,about 1550 amino acids to about 1600 amino acids, about 1600 amino acidsto about 1950 amino acids, about 1600 amino acids to about 1900 aminoacids, about 1600 amino acids to about 1850 amino acids, about 1600amino acids to about 1800 amino acids, about 1600 amino acids to about1750 amino acids, about 1600 amino acids to about 1700 amino acids,about 1600 amino acids to about 1650 amino acids, about 1650 amino acidsto about 1950 amino acids, about 1650 amino acids to about 1900 aminoacids, about 1650 amino acids to about 1850 amino acids, about 1650amino acids to about 1800 amino acids, about 1650 amino acids to about1750 amino acids, about 1650 amino acids to about 1700 amino acids,about 1700 amino acids to about 1950 amino acids, about 1700 amino acidsto about 1900 amino acids, about 1700 amino acids to about 1850 aminoacids, about 1700 amino acids to about 1800 amino acids, about 1700amino acids to about 1750 amino acids, about 1750 amino acids to about1950 amino acids, about 1750 amino acids to about 1900 amino acids,about 1750 amino acids to about 1850 amino acids, about 1750 amino acidsto about 1800 amino acids, about 1800 amino acids to about 1950 aminoacids, about 1800 amino acids to about 1900 amino acids, about 1800amino acids to about 1850 amino acids, about 1850 amino acids to about1950 amino acids, about 1850 amino acids to about 1900 amino acids,about 1900 amino acids to about 1950 amino acids), wherein the aminoacid sequence of each of the encoded portions may optionally partiallyoverlap with the amino acid sequence of a different one of the encodedportions; no single vector of the at least two different vectors encodesan active otoferlin protein (e.g., a full-length otoferlin protein(e.g., a full-length wildtype otoferlin protein)); and, when introducedinto a mammalian cell, the at least two different vectors undergohomologous recombination with each other, thereby forming a recombinednucleic acid, where the recombined nucleic acid encodes an activeotoferlin protein (e.g., a full-length otoferlin protein). In someembodiments, one of the nucleic acid vectors can include a codingsequence that encodes a portion of an otoferlin protein, where theencoded portion is, e.g., about 900 amino acids to about 1950 aminoacids, about 900 amino acids to about 1900 amino acids, about 900 aminoacids to about 1850 amino acids, about 900 amino acids to about 1800amino acids, about 900 amino acids to about 1750 amino acids, about 900amino acids to about 1700 amino acids, about 900 amino acids to about1650 amino acids, about 900 amino acids to about 1600 amino acids, about900 amino acids to about 1550 amino acids, about 900 amino acids toabout 1500 amino acids, about 900 amino acids to about 1450 amino acids,about 900 amino acids to about 1400 amino acids, about 900 amino acidsto about 1350 amino acids, about 900 amino acids to about 1300 aminoacids, about 900 amino acids to about 1250 amino acids, about 900 aminoacids to about 1200 amino acids, about 900 amino acids to about 1150amino acids, about 900 amino acids to about 1100 amino acids, about 900amino acids to about 1050 amino acids, about 900 amino acids to about1000 amino acids, about 900 amino acids to about 950 amino acids, about950 amino acids to about 1950 amino acids, about 950 amino acids toabout 1900 amino acids, about 950 amino acids to about 1850 amino acids,about 950 amino acids to about 1800 amino acids, about 950 amino acidsto about 1750 amino acids, about 950 amino acids to about 1700 aminoacids, about 950 amino acids to about 1650 amino acids, about 950 aminoacids to about 1600 amino acids, about 950 amino acids to about 1550amino acids, about 950 amino acids to about 1500 amino acids, about 950amino acids to about 1450 amino acids, about 950 amino acids to about1400 amino acids, about 950 amino acids to about 1350 amino acids, about950 amino acids to about 1300 amino acids, about 950 amino acids toabout 1250 amino acids, about 950 amino acids to about 1200 amino acids,about 950 amino acids to about 1150 amino acids, about 950 amino acidsto about 1100 amino acids, about 950 amino acids to about 1050 aminoacids, about 950 amino acids to about 1000 amino acids, about 1000 aminoacids to about 1950 amino acids, about 1000 amino acids to about 1900amino acids, about 1000 amino acids to about 1850 amino acids, about1000 amino acids to about 1800 amino acids, about 1000 amino acids toabout 1750 amino acids, about 1000 amino acids to about 1700 aminoacids, about 1000 amino acids to about 1650 amino acids, about 1000amino acids to about 1600 amino acids, about 1000 amino acids to about1550 amino acids, about 1000 amino acids to about 1500 amino acids,about 1000 amino acids to about 1450 amino acids, about 1000 amino acidsto about 1400 amino acids, about 1000 amino acids to about 1350 aminoacids, about 1000 amino acids to about 1300 amino acids, about 1000amino acids to about 1250 amino acids, about 1000 amino acids to about1200 amino acids, about 1000 amino acids to about 1150 amino acids,about 1000 amino acids to about 1100 amino acids, about 1000 amino acidsto about 1050 amino acids, about 1050 amino acids to about 1950 aminoacids, about 1050 amino acids to about 1900 amino acids, about 1050amino acids to about 1850 amino acids, about 1050 amino acids to about1800 amino acids, about 1050 amino acids to about 1750 amino acids,about 1050 amino acids to about 1700 amino acids, about 1050 amino acidsto about 1650 amino acids, about 1050 amino acids to about 1600 aminoacids, about 1050 amino acids to about 1550 amino acids, about 1050amino acids to about 1500 amino acids, about 1050 amino acids to about1450 amino acids, about 1050 amino acids to about 1400 amino acids,about 1050 amino acids to about 1350 amino acids, about 1050 amino acidsto about 1300 amino acids, about 1050 amino acids to about 1250 aminoacids, about 1050 amino acids to about 1200 amino acids, about 1050amino acids to about 1150 amino acids, about 1050 amino acids to about1100 amino acids, about 1100 amino acids to about 1950 amino acids,about 1100 amino acids to about 1900 amino acids, about 1100 amino acidsto about 1850 amino acids, about 1100 amino acids to about 1800 aminoacids, about 1100 amino acids to about 1750 amino acids, about 1100amino acids to about 1700 amino acids, about 1100 amino acids to about1650 amino acids, about 1100 amino acids to about 1600 amino acids,about 1100 amino acids to about 1550 amino acids, about 1100 amino acidsto about 1500 amino acids, about 1100 amino acids to about 1450 aminoacids, about 1100 amino acids to about 1400 amino acids, about 1100amino acids to about 1350 amino acids, about 1100 amino acids to about1300 amino acids, about 1100 amino acids to about 1250 amino acids,about 1100 amino acids to about 1200 amino acids, about 1100 amino acidsto about 1150 amino acids, about 1150 amino acids to about 1950 aminoacids, about 1150 amino acids to about 1900 amino acids, about 1150amino acids to about 1850 amino acids, about 1150 amino acids to about1800 amino acids, about 1150 amino acids to about 1750 amino acids,about 1150 amino acids to about 1700 amino acids, about 1150 amino acidsto about 1650 amino acids, about 1150 amino acids to about 1600 aminoacids, about 1150 amino acids to about 1550 amino acids, about 1150amino acids to about 1500 amino acids, about 1150 amino acids to about1450 amino acids, about 1150 amino acids to about 1400 amino acids,about 1150 amino acids to about 1350 amino acids, about 1150 amino acidsto about 1300 amino acids, about 1150 amino acids to about 1250 aminoacids, about 1150 amino acids to about 1200 amino acids, about 1200amino acids to about 1950 amino acids, about 1200 amino acids to about1900 amino acids, about 1200 amino acids to about 1850 amino acids,about 1200 amino acids to about 1800 amino acids, about 1200 amino acidsto about 1750 amino acids, about 1200 amino acids to about 1700 aminoacids, about 1200 amino acids to about 1650 amino acids, about 1200amino acids to about 1600 amino acids, about 1200 amino acids to about1550 amino acids, about 1200 amino acids to about 1500 amino acids,about 1200 amino acids to about 1450 amino acids, about 1200 amino acidsto about 1400 amino acids, about 1200 amino acids to about 1350 aminoacids, about 1200 amino acids to about 1300 amino acids, about 1200amino acids to about 1250 amino acids, about 1250 amino acids to about1950 amino acids, about 1250 amino acids to about 1900 amino acids,about 1250 amino acids to about 1850 amino acids, about 1250 amino acidsto about 1800 amino acids, about 1250 amino acids to about 1750 aminoacids, about 1250 amino acids to about 1700 amino acids, about 1250amino acids to about 1650 amino acids, about 1250 amino acids to about1600 amino acids, about 1250 amino acids to about 1550 amino acids,about 1250 amino acids to about 1500 amino acids, about 1250 amino acidsto about 1450 amino acids, about 1250 amino acids to about 1400 aminoacids, about 1250 amino acids to about 1350 amino acids, about 1250amino acids to about 1300 amino acids, about 1300 amino acids to about1950 amino acids, about 1300 amino acids to about 1900 amino acids,about 1300 amino acids to about 1850 amino acids, about 1300 amino acidsto about 1800 amino acids, about 1300 amino acids to about 1750 aminoacids, about 1300 amino acids to about 1700 amino acids, about 1300amino acids to about 1650 amino acids, about 1300 amino acids to about1600 amino acids, about 1300 amino acids to about 1550 amino acids,about 1300 amino acids to about 1500 amino acids, about 1300 amino acidsto about 1450 amino acids, about 1300 amino acids to about 1400 aminoacids, about 1300 amino acids to about 1350 amino acids, about 1350amino acids to about 1950 amino acids, about 1350 amino acids to about1900 amino acids, about 1350 amino acids to about 1850 amino acids,about 1350 amino acids to about 1800 amino acids, about 1350 amino acidsto about 1750 amino acids, about 1350 amino acids to about 1700 aminoacids, about 1350 amino acids to about 1650 amino acids, about 1350amino acids to about 1600 amino acids, about 1350 amino acids to about1550 amino acids, about 1350 amino acids to about 1500 amino acids,about 1350 amino acids to about 1450 amino acids, about 1350 amino acidsto about 1400 amino acids, about 1400 amino acids to about 1950 aminoacids, about 1400 amino acids to about 1900 amino acids, about 1400amino acids to about 1850 amino acids, about 1400 amino acids to about1800 amino acids, about 1400 amino acids to about 1750 amino acids,about 1400 amino acids to about 1700 amino acids, about 1400 amino acidsto about 1650 amino acids, about 1400 amino acids to about 1600 aminoacids, about 1400 amino acids to about 1550 amino acids, about 1400amino acids to about 1500 amino acids, about 1400 amino acids to about1450 amino acids, about 1450 amino acids to about 1950 amino acids,about 1450 amino acids to about 1900 amino acids, about 1450 amino acidsto about 1850 amino acids, about 1450 amino acids to about 1800 aminoacids, about 1450 amino acids to about 1750 amino acids, about 1450amino acids to about 1700 amino acids, about 1450 amino acids to about1650 amino acids, about 1450 amino acids to about 1600 amino acids,about 1450 amino acids to about 1550 amino acids, about 1450 amino acidsto about 1500 amino acids, about 1500 amino acids to about 1950 aminoacids, about 1500 amino acids to about 1900 amino acids, about 1500amino acids to about 1850 amino acids, about 1500 amino acids to about1800 amino acids, about 1500 amino acids to about 1750 amino acids,about 1500 amino acids to about 1700 amino acids, about 1500 amino acidsto about 1650 amino acids, about 1500 amino acids to about 1600 aminoacids, about 1500 amino acids to about 1550 amino acids, about 1550amino acids to about 1950 amino acids, about 1550 amino acids to about1900 amino acids, about 1550 amino acids to about 1850 amino acids,about 1550 amino acids to about 1800 amino acids, about 1550 amino acidsto about 1750 amino acids, about 1550 amino acids to about 1700 aminoacids, about 1550 amino acids to about 1650 amino acids, about 1550amino acids to about 1600 amino acids, about 1600 amino acids to about1950 amino acids, about 1600 amino acids to about 1900 amino acids,about 1600 amino acids to about 1850 amino acids, about 1600 amino acidsto about 1800 amino acids, about 1600 amino acids to about 1750 aminoacids, about 1600 amino acids to about 1700 amino acids, about 1600amino acids to about 1650 amino acids, about 1650 amino acids to about1950 amino acids, about 1650 amino acids to about 1900 amino acids,about 1650 amino acids to about 1850 amino acids, about 1650 amino acidsto about 1800 amino acids, about 1650 amino acids to about 1750 aminoacids, about 1650 amino acids to about 1700 amino acids, about 1700amino acids to about 1950 amino acids, about 1700 amino acids to about1900 amino acids, about 1700 amino acids to about 1850 amino acids,about 1700 amino acids to about 1800 amino acids, about 1700 amino acidsto about 1750 amino acids, about 1750 amino acids to about 1950 aminoacids, about 1750 amino acids to about 1900 amino acids, about 1750amino acids to about 1850 amino acids, about 1750 amino acids to about1800 amino acids, about 1800 amino acids to about 1950 amino acids,about 1800 amino acids to about 1900 amino acids, about 1800 amino acidsto about 1850 amino acids, about 1850 amino acids to about 1950 aminoacids, about 1850 amino acids to about 1900 amino acids, or about 1900amino acids to about 1950 amino acids in length.

In some embodiments of these compositions, at least one of the codingsequences includes a nucleotide sequence spanning two neighboring exonsof otoferlin genomic DNA, and lacks the intronic sequence that naturallyoccurs between the two neighboring exons.

In some embodiments, the amino acid sequence of none of the encodedportions overlaps even in part with the amino acid sequence of adifferent one of the encoded portions. In some embodiments, the aminoacid sequence of one or more of the encoded portions partially overlapswith the amino acid sequence of a different one of the encoded portions.In some embodiments, the amino acid sequence of each of the encodedportions partially overlaps with the amino acid sequence of a differentone of the encoded portions.

In some embodiments, the overlapping amino acid sequence is betweenabout 30 amino acid residues to about 1000 amino acids (e.g., or any ofthe subranges of this range described herein) in length.

In some examples, the vectors include two different vectors, each ofwhich comprises not only exon(s), but also a different segment of anintron, wherein the intron includes the nucleotide sequence of an intronthat is present in an otoferlin genomic DNA (e.g., any of the exemplaryintrons in SEQ ID NO: 12 described herein), and wherein the twodifferent segments overlap in sequence by at least 100 nucleotides(e.g., about 100 nucleotides to about 5,000 nucleotides, about 100nucleotides to about 4,500 nucleotides, about 100 nucleotides to about4,000 nucleotides, about 100 nucleotides to about 3,500 nucleotides,about 100 nucleotides to about 3,000 nucleotides, about 100 nucleotidesto about 2,500 nucleotides, about 100 nucleotides to about 2,000nucleotides, about 100 nucleotides to about 1,500 nucleotides, about 100nucleotides to about 1,000 nucleotides, about 100 nucleotides to about800 nucleotides, about 100 nucleotides to about 600 nucleotides, about100 nucleotides to about 400 nucleotides, about 100 nucleotides to about200 nucleotides, about 200 nucleotides to about 5,000 nucleotides, about200 nucleotides to about 4,500 nucleotides, about 200 nucleotides toabout 4,000 nucleotides, about 200 nucleotides to about 3,500nucleotides, about 200 nucleotides to about 3,000 nucleotides, about 200nucleotides to about 2,500 nucleotides, about 200 nucleotides to about2,000 nucleotides, about 200 nucleotides to about 1,500 nucleotides,about 200 nucleotides to about 1,000 nucleotides, about 200 nucleotidesto about 800 nucleotides, about 200 nucleotides to about 600nucleotides, about 200 nucleotides to about 400 nucleotides, about 400nucleotides to about 5,000 nucleotides, about 400 nucleotides to about4,500 nucleotides, about 400 nucleotides to about 4,000 nucleotides,about 400 nucleotides to about 3,500 nucleotides, about 400 nucleotidesto about 3,000 nucleotides, about 400 nucleotides to about 2,500nucleotides, about 400 nucleotides to about 2,000 nucleotides, about 400nucleotides to about 1,500 nucleotides, about 400 nucleotides to about1,000 nucleotides, about 400 nucleotides to about 800 nucleotides, about400 nucleotides to about 600 nucleotides, about 600 nucleotides to about5,000 nucleotides, about 600 nucleotides to about 4,500 nucleotides,about 600 nucleotides to about 4,000 nucleotides, about 600 nucleotidesto about 3,500 nucleotides, about 600 nucleotides to about 3,000nucleotides, about 600 nucleotides to about 2,500 nucleotides, about 600nucleotides to about 2,000 nucleotides, about 600 nucleotides to about1,500 nucleotides, about 600 nucleotides to about 1,000 nucleotides,about 600 nucleotides to about 800 nucleotides, about 800 nucleotides toabout 5,000 nucleotides, about 800 nucleotides to about 4,500nucleotides, about 800 nucleotides to about 4,000 nucleotides, about 800nucleotides to about 3,500 nucleotides, about 800 nucleotides to about3,000 nucleotides, about 800 nucleotides to about 2,500 nucleotides,about 800 nucleotides to about 2,000 nucleotides, about 800 nucleotidesto about 1,500 nucleotides, about 800 nucleotides to about 1,000nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about1,000 nucleotides to about 4,500 nucleotides, about 1,000 nucleotides toabout 4,000 nucleotides, about 1,000 nucleotides to about 3,500nucleotides, about 1,000 nucleotides to about 3,000 nucleotides, about1,000 nucleotides to about 2,500 nucleotides, about 1,000 nucleotides toabout 2,000 nucleotides, about 1,000 nucleotides to about 1,500nucleotides, about 1,500 nucleotides to about 5,000 nucleotides, about1,500 nucleotides to about 4,500 nucleotides, about 1,500 nucleotides toabout 4,000 nucleotides, about 1,500 nucleotides to about 3,500nucleotides, about 1,500 nucleotides to about 3,000 nucleotides, about1,500 nucleotides to about 2,500 nucleotides, about 1,500 nucleotides toabout 2,000 nucleotides, about 2,000 nucleotides to about 5,000nucleotides, about 2,000 nucleotides to about 4,500 nucleotides, about2,000 nucleotides to about 4,000 nucleotides, about 2,000 nucleotides toabout 3,500 nucleotides, about 2,000 nucleotides to about 3,000nucleotides, about 2,000 nucleotides to about 2,500 nucleotides, about2,500 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides toabout 4,500 nucleotides, about 2,500 nucleotides to about 4,000nucleotides, about 2,500 nucleotides to about 3,500 nucleotides, about2,500 nucleotides to about 3,000 nucleotides, about 3,000 nucleotides toabout 5,000 nucleotides, about 3,000 nucleotides to about 4,500nucleotides, about 3,000 nucleotides to about 4,000 nucleotides, about3,000 nucleotides to about 3,500 nucleotides, about 3,500 nucleotides toabout 5,000 nucleotides, about 3,500 nucleotides to about 4,500nucleotides, about 3,500 nucleotides to about 4,000 nucleotides, about4,000 nucleotides to about 5,000 nucleotides, about 4,000 nucleotides toabout 4,500 nucleotides, about 4,500 nucleotides to about 5,000nucleotides), in length.

The overlapping nucleotide sequence in any two of the different vectorscan include part or all of one or more exons of an otoferlin gene (e.g.,any one or more of the exemplary exons in SEQ ID NO: 12 describedherein).

In some embodiments, the number of different vectors in the compositionis two, three, four, or five. In compositions where the number ofdifferent vectors in the composition is two, the first of the twodifferent vectors can include a coding sequence that encodes anN-terminal portion of the otoferlin protein. In some examples, theN-terminal portion of the otoferlin gene is between about 30 amino acidsto about 1950 amino acids (or any of the subranges of this rangedescribed above) in length. In some examples, the first vector furtherincludes one or both of a promoter (e.g., any of the promoters describedherein or known in the art) and a Kozak sequence (e.g., any of theexemplary Kozak sequences described herein or known in the art). In someexamples, the first vector includes a promoter that is an induciblepromoter, a constitutive promoter, or a tissue-specific promoter. Insome examples, the second of the two different vectors includes a codingsequence that encodes a C-terminal portion of the otoferlin protein. Insome examples, the C-terminal portion of the otoferlin protein isbetween 30 amino acids to about 1950 amino acids (or any of thesubranges of this range described above) in length. In some examples,the second vector further includes a poly(A) signal sequence.

In some examples where the number of different vectors in thecomposition is two, the N-terminal portion encoded by one of the twovectors can include a portion comprising amino acid position 1 to aboutamino acid position 1,950, about amino acid position 1,940, about aminoacid position 1,930, about amino acid position 1,920, about amino acidposition 1,910, about amino acid position 1,900, about amino acidposition 1,900, about amino acid position 1,890, about amino acidposition 1,880, about amino acid position 1,870, about amino acidposition 1,860, about amino acid position 1,850, about amino acidposition 1,840, about amino acid position 1,830, about amino acidposition 1,820, about amino acid position 1,810, about amino acidposition 1,800, about amino acid position 1,790, about amino acidposition 1,780, about amino acid position 1,770, about amino acidposition 1,760, about amino acid position 1,750, about amino acidposition 1,740, about amino acid position 1,730, about amino acidposition 1,720, about amino acid position 1,710, about amino acidposition 1,700, about amino acid position, about amino acid position1,690, about amino acid position 1,680, about amino acid position 1,670,about amino acid position 1,660, about amino acid position 1,650, aboutamino acid position 1,640, about amino acid position 1,630, about aminoacid position 1,620, about amino acid position 1,610, about amino acidposition 1,600, about amino acid position 1,590, about amino acidposition 1,580, about amino acid position 1,570, about amino acidposition 1,560, about amino acid position 1,550, about amino acidposition 1,540, about amino acid position 1,530, about amino acidposition 1,520, about amino acid position 1,510, amino acid position 1to about amino acid position 1,500, about amino acid position 1,490,about amino acid position 1,480, about amino acid position 1,470, aboutamino acid position 1,460, about amino acid position 1,450, about aminoacid position 1,440, about amino acid position 1,430, about amino acidposition 1,420, about amino acid position 1,410, about amino acidposition 1,400, about amino acid position 1,390, about amino acidposition 1,380, about amino acid position 1,370, about amino acidposition 1,360, about amino acid position 1,350, about amino acidposition 1,340, about amino acid position 1,330, about amino acidposition 1,320, about amino acid position 1,310, about amino acidposition 1,300, about amino acid position 1,290, about amino acidposition 1,280, about amino acid position 1,270, about amino acidposition 1,260, about amino acid position 1,250, about amino acidposition 1,240, about amino acid position 1,230, about amino acidposition 1,220, about amino acid position 1,210, about amino acidposition 1,200, about amino acid position 1,190, about amino acidposition 1,180, about amino acid position 1,170, about amino acidposition 1,160, about amino acid position 1,150, about amino acidposition 1,140, about amino acid position 1,130, about amino acidposition 1,120, about amino acid position 1,110, about amino acidposition 1,100, about amino acid position 1,090, about amino acidposition 1,080, about amino acid position 1,070, about amino acidposition 1,060, about amino acid position 1,050, about amino acidposition 1,040, about amino acid position 1,030, about amino acidposition 1,020, about amino acid position 1,010, about amino acidposition 1,000, about amino acid position 990, about amino acid position980, about amino acid position 970, about amino acid position 960, aboutamino acid position 950, about amino acid position 940, about amino acidposition 930, about amino acid position 920, about amino acid position910, about amino acid position 900, about amino acid position 890, aboutamino acid position 880, about amino acid position 870, about amino acidposition 860, about amino acid position 850, about amino acid position840, about amino acid position 830, about amino acid position 820, aboutamino acid position 810, about amino acid position 800, about amino acidposition 790, about amino acid position 780, about amino acid position770, about amino acid position 760, about amino acid position 750, aboutamino acid position 740, about amino acid position 730, about amino acidposition 720, about amino acid position 710, about amino acid position700, about amino acid position 690, about amino acid position 680, aboutamino acid position 670, about amino acid position 660, about amino acidposition 650, about amino acid position 640, about amino acid position630, about amino acid position 620, about amino acid position 610, aboutamino acid position 600, about amino acid position 590, about amino acidposition 580, about amino acid position 570, about amino acid position560, about amino acid position 550, about amino acid position 540, aboutamino acid position 530, about amino acid position 520, about amino acidposition 510, about amino acid position 500, about amino acid position490, about amino acid position 480, about amino acid position 470, aboutamino acid position 460, about amino acid position 450, about amino acidposition 440, about amino acid position 430, about amino acid position420, about amino acid position 410, about amino acid position 400, aboutamino acid position 390, about amino acid position 380, about amino acidposition 370, about amino acid position 360, about amino acid position350, about amino acid position 340, about amino acid position 330, aboutamino acid position 320, about amino acid position 310, about amino acidposition 300, about amino acid position 290, about amino acid position280, about amino acid position 270, about amino acid position 260, aboutamino acid position 250, about amino acid position 240, about amino acidposition 230, about amino acid position 220, about amino acid position210, about amino acid position 200, about amino acid position 190, aboutamino acid position 180, about amino acid position 170, about amino acidposition 160, about amino acid position 150, about amino acid position140, about amino acid position 130, about amino acid position 120, aboutamino acid position 110, about amino acid position 100, about amino acidposition 90, about amino acid position 80, about amino acid position 70,about amino acid position 60, about amino acid position 50, or aboutamino acid position 40 of a wildtype otoferlin protein (e.g., SEQ ID NO:5).

In some examples where the number of different vectors in thecomposition is two, the N-terminal portion of the precursor otoferlinprotein can include a portion comprising amino acid position 1 to aminoacid position 310, amino acid position 1 to about amino acid position320, amino acid position 1 to about amino acid position 330, amino acidposition 1 to about amino acid position 340, amino acid position 1 toabout amino acid position 350, amino acid position 1 to about amino acidposition 360, amino acid position 1 to about amino acid position 370,amino acid position 1 to about amino acid position 380, amino acidposition 1 to about amino acid position 390, amino acid position 1 toabout amino acid position 400, amino acid position 1 to about amino acidposition 410, amino acid position 1 to about amino acid position 420,amino acid position 1 to about amino acid position 430, amino acidposition 1 to about amino acid position 440, amino acid position 1 toabout amino acid position 450, amino acid position 1 to about amino acidposition 460, amino acid position 1 to about amino acid position 470,amino acid position 1 to about amino acid position 480, amino acidposition 1 to about amino acid position 490, amino acid position 1 toabout amino acid position 500, amino acid position 1 to about amino acidposition 510, amino acid position 1 to about amino acid position 520,amino acid position 1 to about amino acid position 530, amino acidposition 1 to about amino acid position 540, amino acid position 1 toabout amino acid position 550, amino acid position 1 to about amino acidposition 560, amino acid position 1 to about amino acid position 570,amino acid position 1 to about amino acid position 580, amino acidposition 1 to about amino acid position 590, amino acid position 1 toabout amino acid position 600, amino acid position 1 to about amino acidposition 610, amino acid position 1 to about amino acid position 620,amino acid position 1 to about amino acid position 630, amino acidposition 1 to about amino acid position 640, amino acid position 1 toabout amino acid position 650, amino acid position 1 to about amino acidposition 660, amino acid position 1 to about amino acid position 670,amino acid position 1 to about amino acid position 680, amino acidposition 1 to about amino acid position 690, amino acid position 1 toabout amino acid position 700, amino acid position 1 to about amino acidposition 710, amino acid position 1 to about amino acid position 720,amino acid position 1 to about amino acid position 730, amino acidposition 1 to about amino acid position 740, amino acid position 1 toabout amino acid position 750, amino acid position 1 to about amino acidposition 760, amino acid position 1 to about amino acid position 770,amino acid position 1 to about amino acid position 780, amino acidposition 1 to about amino acid position 790, amino acid position 1 toabout amino acid position 800, amino acid position 1 to about amino acidposition 810, amino acid position 1 to about amino acid position 820,amino acid position 1 to about amino acid position 830, amino acidposition 1 to about amino acid position 840, amino acid position 1 toabout amino acid position 850, amino acid position 1 to about amino acidposition 860, amino acid position 1 to about amino acid position 870,amino acid position 1 to about amino acid position 880, amino acidposition 1 to about amino acid position 890, amino acid position 1 toabout amino acid position 900, amino acid position 1 to about amino acidposition 910, amino acid position 1 to about amino acid position 920,amino acid position 1 to about amino acid position 930, amino acidposition 1 to about amino acid position 940, amino acid position 1 toabout amino acid position 950, amino acid position 1 to about amino acidposition 960, amino acid position 1 to about amino acid position 970,amino acid position 1 to about amino acid position 980, amino acidposition 1 to about amino acid position 990, amino acid position 1 toabout amino acid position 1,000, amino acid position 1 to about aminoacid position 1,010, amino acid position 1 to about amino acid position1,020, amino acid position 1 to about amino acid position 1,030, aminoacid position 1 to about amino acid position 1,040, amino acid position1 to about amino acid position 1,050, amino acid position 1 to aboutamino acid position 1,060, amino acid position 1 to about amino acidposition 1,070, amino acid position 1 to about amino acid position1,080, amino acid position 1 to about amino acid position 1,090, aminoacid position 1 to about amino acid position 1,100, amino acid position1 to about amino acid position 1,110, amino acid position 1 to aboutamino acid position 1,120, amino acid position 1 to about amino acidposition 1,130, amino acid position 1 to about amino acid position1,140, amino acid position 1 to about amino acid position 1,150, aminoacid position 1 to about amino acid position 1,160, amino acid position1 to about amino acid position 1,170, amino acid position 1 to aboutamino acid position 1,180, amino acid position 1 to about amino acidposition 1,190, amino acid position 1 to about amino acid position1,200, amino acid position 1 to about amino acid position 1,210, aminoacid position 1 to about amino acid position 1,220, amino acid position1 to about amino acid position 1,230, amino acid position 1 to aboutamino acid position 1,240, amino acid position 1 to about amino acidposition 1,250, amino acid position 1 to about amino acid position1,260, amino acid position 1 to about amino acid position 1,270, aminoacid position 1 to about amino acid position 1,280, amino acid position1 to about amino acid position 1,290, amino acid position 1 to aboutamino acid position 1,300, amino acid position 1 to about amino acidposition 1,310, amino acid position 1 to about amino acid position1,320, amino acid position 1 to about amino acid position 1,330, aminoacid position 1 to about amino acid position 1,340, amino acid position1 to about amino acid position 1,350, amino acid position 1 to aboutamino acid position 1,360, amino acid position 1 to about amino acidposition 1,370, amino acid position 1 to about amino acid position1,380, amino acid position 1 to about amino acid position 1,390, aminoacid position 1 to about amino acid position 1,400, amino acid position1 to about amino acid position 1,410, amino acid position 1 to aboutamino acid position 1,420, amino acid position 1 to about amino acidposition 1,430, amino acid position 1 to about amino acid position1,440, amino acid position 1 to about amino acid position 1,450, aminoacid position 1 to about amino acid position 1,460, amino acid position1 to about amino acid position 1,470, amino acid position 1 to aboutamino acid position 1,480, amino acid position 1 to about amino acidposition 1,490, amino acid position 1 to about amino acid position1,500, amino acid position 1 to about amino acid position 1,510, aminoacid position 1 to about amino acid position 1,520, amino acid position1 to about amino acid position 1,530, amino acid position 1 to aboutamino acid position 1,540, amino acid position 1 to about amino acidposition 1,550, amino acid position 1 to about amino acid position1,560, amino acid position 1 to about amino acid position 1,570, aminoacid position 1 to about amino acid position 1,580, amino acid position1 to about amino acid position 1,590, amino acid position 1 to aboutamino acid position 1,600, amino acid position 1 to about amino acid1,610, amino acid position 1 to about amino acid 1,620, amino acidposition 1 to about amino acid 1,630, amino acid position 1 to aboutamino acid 1,640, amino acid position 1 to about amino acid 1,650, aminoacid position 1 to about amino acid 1,660, amino acid position 1 toabout amino acid 1,670, amino acid position 1 to about amino acid 1,680,amino acid position 1 to about amino acid 1,690, amino acid position 1to about amino acid 1,700, amino acid position 1 to about amino acid1,710, amino acid position 1 to about amino acid 1,720, amino acidposition 1 to about amino acid 1,730, amino acid position 1 to aboutamino acid 1,740, amino acid position 1 to about amino acid 1,750, aminoacid position 1 to about amino acid 1,760, amino acid position 1 toabout amino acid 1,770, amino acid position 1 to about amino acid 1,780,amino acid position 1 to about amino acid 1,790, amino acid position 1to about amino acid 1,800, amino acid position 1 to about amino acid1,810, amino acid position 1 to about amino acid 1,820, amino acidposition 1 to about amino acid 1,830, amino acid position 1 to aboutamino acid 1,840, amino acid position 1 to about amino acid 1,850, aminoacid position 1 to about amino acid 1,860, amino acid position 1 toabout amino acid 1,870, amino acid position 1 to about amino acid 1,880,amino acid position 1 to about amino acid 1,890, amino acid position 1to about amino acid 1,900, amino acid position 1 to about amino acid1,910, amino acid position 1 to about amino acid 1,920, amino acidposition 1 to about amino acid 1,930, amino acid position 1 to aboutamino acid 1,940, amino acid position 1 to about amino acid 1,950, aminoacid position 1 to about amino acid 1,960, amino acid position 1 toabout amino acid 1,970, amino acid position 1 to about amino acid 1,980of a wildtype otoferlin protein (e.g., SEQ ID NO: 5).

In some examples, the composition includes two vectors, where a first ofthe two vectors includes a coding sequence that is at least 80%, atleast 82%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 98%, at least 99%, or100% identical to SEQ ID NO: 62, and the second of the two vectorsincludes a coding sequence that is at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQID NO: 63.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., that is capable of replication when associated withthe proper control elements and that can transfer gene sequences betweencells. Thus, the term includes cloning and expression vehicles, as wellas viral vectors. In some embodiments, useful vectors are contemplatedto be those vectors in which the nucleic acid segment to be transcribedis positioned under the transcriptional control of a promoter.

In some examples, a vector can be an artificial chromosome (e.g., ahuman artificial chromosome (HAC), a yeast artificial chromosome (YAC),a bacterial artificial chromosome (BAC), or a P1-derived artificialchromosome (PAC)) or a viral vector (e.g., any adenoviral vectors (e.g.,pSV or pCMV vectors), any retroviral vectors as described herein, andany Gateway® vectors). A vector can, e.g., include sufficient cis-actingelements for expression; other elements for expression can be suppliedby the host mammalian cell or in an in vitro expression system.

A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell, or introduced synthetic machinery, required toinitiate the specific transcription of a polynucleotide sequence (e.g.,a gene). The phrases “operatively positioned,” “under control” or “undertranscriptional control” means that the promoter is in the correctlocation and orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the gene. The term “expressionvector or construct” means any type of genetic construct containing anucleic acid in which part or all of the nucleic acid coding sequence iscapable of being transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA, miRNA inhibitor) from a transcribed gene.

Vectors include all those known in the art, including cosmids, plasmids(e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses,retroviruses, adenoviruses, and adeno-associated viruses) thatincorporate the recombinant polynucleotide. Skilled practitioners willbe capable of selecting suitable vectors and mammalian cells for makingany of the nucleic acids described herein.

In some embodiments the vector is a plasmid (i.e. a circular DNAmolecule that can autonomously replicate inside a cell). In someembodiments, the vector can be a cosmid (e.g., pWE and sCos series (Wahlet al. (1987) Proc. Natl. Acad. Sci. USA 84:2160-2164, Evans et al.(1989) Proc. Natl. Acad. Sci. USA 86:5030-5034).

The term “transfer vector” refers to a composition of matter whichincludes an isolated nucleic acid and which can be used to deliver theisolated nucleic acid to the interior of a cell. Numerous vectors areknown in the art including, but not limited to, linear polynucleotides,polynucleotides associated with ionic or amphiphilic compounds,plasmids, and viruses. Thus, the term “transfer vector” includes anautonomously replicating plasmid or a virus. The term should also beconstrued to further include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into cells, such as, for example, apolylysine compound, liposome, and the like. Examples of viral transfervectors include, but are not limited to, adenoviral vectors,adeno-associated virus vectors, retroviral vectors, lentiviral vectors,and the like.

The term “expression vector” refers to a vector including a recombinantpolynucleotide including expression control sequences operatively linkedto a nucleotide sequence to be expressed. An expression vector includessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, includingcosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

In some embodiments, the vector(s) is an artificial chromosome. Anartificial chromosome is a genetically engineered chromosome that can beused as a vector to carry large DNA inserts. In some embodiments, theartificial chromosome is human artificial chromosome (HAC) (see, e.g.,Kouprina et al., Expert Opin. DrugDeliv 11(4): 517-535, 2014; Basu etal., Pediatr. Clin. North Am. 53: 843-853, 2006; Ren et al., Stem. CellRev. 2(1):43-50, 2006; Kazuki et al., Mol. Ther. 19(9):1591-1601, 2011;Kazuki et al., Gen. Ther. 18: 384-393, 2011; and Katoh et al., Biochem.Biophys. Res. Commun. 321:280-290, 2004).

In some embodiments, the vector(s) is a yeast artificial chromosome(YAC) (see, e.g., Murray et al., Nature 305: 189-193, 1983; Ikeno et al.(1998) Nat. Biotech. 16:431-439, 1998). In some embodiments, thevector(s) is a bacterial artificial chromosome (BAC) (e.g., pBeloBAC11,pECBAC1, and pBAC108L). In some embodiments, the vector(s) is aP1-derived artificial chromosome (PAC). Examples of artificialchromosome are known in the art.

In some embodiments, the vector(s) is a viral vector (e.g.,adeno-associated virus, adenovirus, lentivirus, and retrovirus).Non-limiting examples of viral vectors are described herein.

Recombinant AAV Vectors

“Recombinant AAV vectors” or “rAAVs” of the disclosure are typicallycomprised of, at a minimum, a transgene or a portion thereof and aregulatory sequence, and optionally 5′ and 3′ AAV inverted terminalrepeats (ITRs). It is this recombinant AAV vector which is packaged intoa capsid protein and delivered to a selected target cell. In someembodiments, the transgene is a nucleic acid sequence, heterologous tothe vector sequences, which encodes a polypeptide, protein, functionalRNA molecule (e.g., miRNA, miRNA inhibitor) or other gene product, ofinterest. The nucleic acid coding sequence is operatively linked toregulatory components in a manner which permits transgene transcription,translation, and/or expression in a cell of a target tissue.

In some embodiments, the vector(s) is an adeno-associated viral vector(AAV) (see, e.g., Asokan et al., Mol. Ther. 20: 699-7080, 2012).“Recombinant AAV vectors” or “rAAVs” are typically composed of, at aminimum, a transgene or a portion thereof and a regulatory sequence, andoptionally 5′ and 3′ AAV inverted terminal repeats (ITRs). Such arecombinant AAV vector is packaged into a capsid to form an rAAVparticle and delivered to a selected target cell (e.g., an inner haircell).

ITRs

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 nt in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al. “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present disclosure is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types. Insome embodiments, an ITR is or comprises 145 nucleotides. In someembodiments an ITR is a wild-type AAV2 ITR, e.g., the 5′ ITR of SEQ IDNO: 97 and the 3′ ITR of SEQ ID NO: 104. In some embodiments an ITR isderived from a wild-type AAV2 ITR and includes one or moremodifications, e.g., truncations, deletions, substitutions or insertionsas is known in the art. In some embodiments, an ITR comprises fewer than145 nucleotides, e.g., 127, 130, 134 or 141 nucleotides. For example, insome embodiments, an ITR comprises 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or145 nucleotides.

A non-limiting example of a 5′ AAV ITR sequence is SEQ ID NO: 59. Anon-limiting example of a 3′ AAV ITR sequence is SEQ ID NO: 60. In someembodiments, vectors and/or constructs of the present disclosurecomprise a 5′ AAV ITR and/or a 3′ AAV ITR. In some embodiments, a 5′ AAVITR sequence is SEQ ID NO: 97. In some embodiments, a 3′ AAV ITRsequence is SEQ ID NO: 104. In some embodiments, the 5′ AAV ITR sequenceis SEQ ID NO: 97 and the 3′ AAV ITR sequence is SEQ ID NO: 104. In someembodiments, the 5′ and a 3′ AAV ITRs (e.g., SEQ ID NOs: 97 and 104)flank a portion of a transgene and/or construct comprising a portion ofOTOF (e.g., SEQ ID NO: 101 or 107).

Other Elements

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elements thatare operably linked to the transgene in a manner which permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by thedisclosure. As used herein, “operably linked” sequences include bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest.

AAV vectors as described herein may include any of the regulatoryelements described herein (e.g., one or more of a promoter, a polyAsequence, and an IRES).

Capsids

In some embodiments, one or more recombinant AAV vectors of the presentdisclosure is packaged into a capsid of the AAV2, 3, 4, 5, 6, 7, 8, 9,10, rh8, rh10, rh39, rh43 or Anc80 serotype or one or more hybridsthereof. In some embodiments, a capsid is from an ancestral serotype.For example, in some embodiments, the capsid is an Anc80 capsid (e.g.,an Anc80L65 capsid). In some embodiments, the capsid comprises apolypeptide represented by SEQ ID NO: 109. In some embodiments, thecapsid comprises a polypeptide with at least 85%, 90%, 95%, 98% or 99%sequence identity to the polypeptide represented by SEQ ID NO: 109.

Any combination of ITRs and capsids may be used in recombinant AAVvectors of the present disclosure, for example, wild-type or variantAAV2 ITRs and Anc80 capsid, wild-type or variant AAV2 ITRs and AAV6capsid, etc. In some embodiments of the present disclosure an rAAVparticle is an rAAV2/Anc80 particle which comprises an Anc80 capsid(e.g., comprising a polypeptide of SEQ ID NO: 109) that encapsidates anucleic acid vector with wild-type AAV2 ITRs (e.g., SEQ ID NOs: 97 and104) flanking a portion of a transgene and/or construct comprising aportion of OTOF (e.g., SEQ ID NO: 101 or 107).

Recombinant AV and Lentiviral Vectors

Also provided are therapeutic compositions including one or moreadenoviral (AV) vectors, wherein the one or the plurality of AV vectorsare capable of constituting an auditory polypeptide messenger RNA in atarget cell of a human subject to whom the therapeutic composition isadministered. Preferably, the one or the plurality of AV vectors arecapable of constituting a full-length auditory polypeptide messenger RNAin a target cell of a human subject to whom the therapeutic compositionis administered.

In some embodiments, the vector(s) is an adenovirus (see, e.g., Dmitrievet al. (1998) J. Virol. 72: 9706-9713; and Poulin et al., J. Virol 8:10074-10086, 2010). In some embodiments, the vector(s) is a retrovirus(see, e.g., Maier et al. (2010) Future Microbiol 5: 1507-23).

In some embodiments, the vector(s) is a lentivirus (see, e.g., Matrai etal. (2010) Mol Ther. 18: 477-490; Banasik et al. (2010) Gene Ther.17:150-7; and Wanisch et al. (2009) Mol. Ther. 17: 1316-32). Alentiviral vector refers to a vector derived from at least a portion ofa lentivirus genome, including especially a self-inactivating lentiviralvector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).Non-limiting lentivirus vectors that may be used in the clinic includethe LENTIVECTOR® gene delivery technology from Oxford BioMedica, theLENTIMAX™ vector system from Lentigen, and the like. Other types oflentiviral vectors are also available and would be known to one skilledin the art.

The term “lentivirus” refers to a genus of the Retroviridae family.Lentiviruses are unique among the retroviruses in being able to infectnon-dividing cells; they can deliver a significant amount of geneticinformation into the DNA of the host cell, so they are one of the mostefficient methods of a gene delivery vector. HIV, SIV, and FIV are allexamples of lentiviruses.

The term “lentiviral vector” refers to a vector derived from at least aportion of a lentivirus genome, including especially a self-inactivatinglentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentivirus vectors that may be usedin the clinic include but are not limited to, e.g., the LENTIVECTOR®.gene delivery technology from Oxford BioMedica, the LENTIMAX™ vectorsystem from Lentigen and the like. Nonclinical types of lentiviralvectors are also available and would be known to one skilled in the art.

For example, provided are therapeutic compositions including one or aplurality of lentiviral vectors, wherein the one or the plurality oflentiviral vectors are capable of constituting an auditory polypeptidemessenger RNA in a target cell of a human subject to whom thetherapeutic composition is administered. In one embodiment, the one orthe plurality of lentiviral vectors are capable of constituting afull-length auditory polypeptide messenger RNA in a target cell of ahuman subject to whom the therapeutic composition is administered.

In some embodiments of any of the compositions described herein, thefirst vector includes an ITR (e.g., any of the exemplary ITR sequencesdescribed herein), a promoter and/or enhancer (e.g., any of theexemplary enhancers and any of the exemplary promoters describedherein), an intron sequence of a OTOF gene (e.g., a human OTOF gene,e.g., any of the exemplary intron sequences of a human OTOF genedescribed herein), a Kozak sequence (e.g., any of the exemplary Kozaksequences described herein), and a sequence encoding a first, N-terminalportion of a human otoferlin protein (e.g., any of the exemplarysequences encoding a first, N-terminal portion of a human otoferlinprotein described herein), an AK sequence (e.g., any of the exemplary AKsequences described herein), and an ITR (e.g., any of the exemplary ITRsequences described herein). In some embodiments of any of thecompositions described herein, the second vector includes an ITRsequence (e.g., any of the exemplary ITR sequences described herein), anAK sequence (e.g., any of the exemplary AK sequences described herein),a splicing acceptor sequence (e.g., any of the splicing acceptorsequences described herein), a sequence encoding a second portion of ahuman otoferlin protein (e.g., any of the exemplary sequences encoding asecond, C-terminal portion of a human otoferlin protein describedherein), a poly(A) signal sequence (e.g., any of the exemplary poly(A)signal sequences described herein), a stuffer sequence (e.g., any of theexemplary stuffer sequences described herein), and an ITR sequence(e.g., any of the exemplary ITR sequences described herein).

In some embodiments of any of the compositions described herein, thevector is pAAV-AK-SA-3′mOTOF-EWB (SEQ ID NO: 39), depicted in FIGS. 11,17 and 56 , or is a vector including a sequence that is at least 80%, atleast 82%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 98%, at least 99%, or100% identical to SEQ ID NO: 39. The pAAV-AK-SA-3′mOTOF-EWB vector is7625 bp in length and has an AK sequence at nucleotide positions 2-78, asplicing acceptor (SA) site at nucleotide positions 79-129, a 3′ mOTOFat nucleotide positions 130-3540, C2D at nucleotide positions 490-891,C2E at nucleotide positions 1996-2516, C2F at nucleotide positions2749-3234, a WPRE at nucleotide positions 3595-4188, an ampicillin (AMP)resistance gene at nucleotide positions 5537-6537, a bovine growthhormone poly A-tail (bGH pA) at nucleotide positions 4212-4422, aphage-derived fl(+) origin of replication (ORI) at nucleotide positions4674-5133, an origin of replication (ORI) at nucleotide positions6787-7012.

In some embodiments of any of the compositions described herein, thevector is pAAV-SA-3′mOTOF-EWB (SEQ ID NO: 40), depicted in FIGS. 12, 20and 54 , or is a vector including a sequence that is at least 80%, atleast 82%, at least 84%, at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 98%, at least 99%, or100% identical to SEQ ID NO: 40. The pAAV-SA-3′mOTOF-EWB vector is 7548bp in length and has a splicing acceptor (SA) site at nucleotidepositions 2-52, a 3′ mOTOF at nucleotide positions 53-3463, C2D atnucleotide positions 413-814, C2E at nucleotide positions 1919-2439, C2Fat nucleotide positions 2672-3157, a WPRE at nucleotide positions3518-4111, an ampicillin (AMP) resistance gene at nucleotide positions5460-6460, a bovine growth hormone poly A-tail (bGH pA) at nucleotidepositions 4135-4345, and a phage-derived fl(+) origin of replication(ORI) at nucleotide positions 4597-5056.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-eGFP-P2A-5′mOTOF-SD (SEQ ID NO: 41), depicted inFIGS. 13, 18 and 53 , or is a vector including a sequence that is atleast 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO: 41. ThepAAV-HBA-eGFP-P2A-5′mOTOF-SD vector is 7346 bp in length and has a Kozaksequence(*) at nucleotide positions 662-667, an enhanced greenfluorescent protein (eGFP) sequence at nucleotide positions 668-1384, aP2A at nucleotide positions 1391-1456, a Kozak sequence at nucleotidepositions 1463-1468, a 5′ mOTOF sequence at nucleotide positions1469-3988, a C2A at nucleotide positions 1469-1831, a C2B at nucleotidepositions 2231-2599, a C2C at nucleotide positions 2720-3091, human OTOFexon 21 at nucleotide positions 3872-3988, a splicing donor (SD) site atnucleotide positions 3989-4070, an AMP resistance gene at nucleotidepositions 5186-6186, a fl(+)ORI at nucleotide positions 4323-4782, anORI at nucleotide positions 6436-6661, and a human cytomegalovirus(hCMV) enhancer at nucleotide positions 7277-272.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-eGFP-P2A-5′mOTOF-SD-AK (SEQ ID NO: 42), depicted inFIGS. 14, 19 and 55 , or is a vector including a sequence that is atleast 80%, at least 82%, at least 84%, at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO: 42. ThepAAV-HBA-eGFP-P2A-5′mOTOF-SD-AK is 7423 bp in length and has a Kozaksequence(*) at nucleotide positions 662-667, an enhanced greenfluorescent protein (eGFP) sequence at nucleotide positions 668-1384, aP2A at nucleotide positions 1391-1456, a Kozak sequence at nucleotidepositions 1463-1468, a 5′ mOTOF sequence at nucleotide positions1469-3988, a C2A at nucleotide positions 1469-1831, a C2B at nucleotidepositions 2231-2599, a C2C at nucleotide positions 2720-3091, human OTOFexon 21 at nucleotide positions 3872-3988, a splicing donor (SD) site atnucleotide positions 3989-4070, an AK sequence at nucleotide positions4071-4147, an AMP resistance gene at nucleotide positions 5263-6263, afl(+)ORI at nucleotide positions 4400-4859, an ORI at nucleotidepositions 6513-6738, and a human cytomegalovirus (hCMV) enhancer atnucleotide positions 7354-272.

In some embodiments of any of the compositions described herein, thevector is pAKOS102 (SEQ ID NO: 43), or is a vector including a sequencethat is at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 92%, at least 94%, at least 96%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 43. pAKOS102 is shownin FIG. 21 . The pAKOS102 vector is 7006 bp in length and has an AAV ITRsequence at nucleotide positions 1-130, a factor VIII stuffer sequenceat nucleotide positions 145-1144, a human cytomegalovirus (hCMV)enhancer at nucleotide positions 1145-1486, a human ACTB promoter atnucleotide positions 1487-1869, a Kozak sequence at nucleotide positions1883-1888, a 5′ hOTOF isoform 5 sequence at nucleotide positions1889-4411, a SD intron sequence at nucleotide positions 4412-4493, anAAV2 ITR sequence at nucleotide positions 4519-4659, a fl(+)ORI atnucleotide positions 4734-5189, an KAN resistance gene at nucleotidepositions 5469-6278, and an ORI at nucleotide positions 6357-6945.

In some embodiments of any of the compositions described herein, thevector is pAKOS104 (SEQ ID NO: 45), or is a vector including a sequencethat is at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 92%, at least 94%, at least 96%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 45. pAKOS104 is shownin FIG. 24 . The pAKOS104 vector is 7083 bp in length and has an AAV ITRsequence at nucleotide positions 1-130, a factor VIII stuffer sequenceat nucleotide positions 145-1144, a human cytomegalovirus (hCMV)enhancer at nucleotide positions 1145-1486, a human ACTB promoter atnucleotide positions 1487-1869, a Kozak sequence at nucleotide positions1883-1888, a 5′ hOTOF isoform 5 sequence at nucleotide positions1889-4411, a SD intron sequence at nucleotide positions 4412-4493, an AKsequence at nucleotide positions 4494-4570, an AAV2 ITR sequence atnucleotide positions 4596-4736, a fl(+)ORI at nucleotide positions4811-5266, an KAN resistance gene at nucleotide positions 5546-6355, andan ORI at nucleotide positions 6434-7022.

In some embodiments of any of the compositions described herein, thevector is pAKOS105_GFP (SEQ ID NO: 48), depicted in FIG. 29 , or is avector including a sequence that is at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQID NO: 48. The pAKOS105_GFP vector is 7761 bp in length and has an AAVITR sequence at nucleotide positions 1-130, an AK sequence at nucleotidepositions 145-221, a SA site sequence at nucleotide positions 222-272, a3′ hOTOF isoform 5 sequence at nucleotide positions 273-3740, a T2Asequence at nucleotide positions 3750-3803, a turboGFP sequence atnucleotide positions 3804-4499, a bGH poly(A) signal at nucleotidepositions 4509-4748, a factor VIII stuffer sequence at nucleotidepositions 4749-5248, an AAV2 ITR sequence at nucleotide positions5274-5414, a fl(+)ORI at nucleotide positions 5489-5944, an KANresistance gene at nucleotide positions 6224-7033, and an ORI atnucleotide positions 7112-7700.

In some embodiments of any of the compositions described herein, thevector is pAKOS109 (SEQ ID NO: 52), or is a vector including a sequencethat is at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 92%, at least 94%, at least 96%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 52. pAKOS109 is shownin FIGS. 36 , The pAKOS109 vector is 7056 bp in length and has an AAVITR sequence at nucleotide positions 1-130, a human cytomegalovirus(hCMV) enhancer at nucleotide positions 145-524, a chicken 3-actinpromoter at nucleotide positions 527-802, a chimeric intron atnucleotide positions 803-1815, a Kozak sequence at nucleotide positions1856-1861, a 5′ hOTOF isoform 5 sequence at nucleotide positions1862-4384, a SD intron sequence at nucleotide positions 4385-4466, an AKsequence at nucleotide positions 4467-4543, an AAV2 ITR sequence atnucleotide positions 4569-4709, a fl(+)ORI at nucleotide positions4784-5239, an KAN resistance gene at nucleotide positions 5519-6328, andan ORI at nucleotide positions 6407-6995.

In some embodiments of any of the compositions described herein, thevector is p109 (SEQ ID NO: 84) or is a vector that includes a sequencethat is at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 92%, at least 94%, at least 96%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 84. The p109 vectoris 4,711 bp in length and is shown in FIG. 38 .

In some embodiments of any of the compositions described herein, thevector is p105 (SEQ ID NO: 85) or is a vector that includes a sequencethat is at least 80%, at least 82%, at least 84%, at least 86%, at least88%, at least 90%, at least 92%, at least 94%, at least 96%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 85. The p105 vectoris 4,664 bp in length and is shown in FIGS. 28 and 39 .

In some embodiments of any of the compositions described herein, thevector is 105.WPRE, shown in FIG. 40 . The WPRE sequence present in the105.WPRE vector is SEQ ID NO: 69.

In some embodiments of any of the compositions described herein, thevector is p108, shown in FIG. 41 . The p108 vector includes the FVIIIstuffer sequence of SEQ ID NO: 58 and the CMV enhancer and promotersequence of SEQ ID NO: 70.

In some embodiments of any of the compositions described herein, thevector is 1OTOF18.CL1, shown in FIG. 42 . The 1OTOF18.CL1 vectorincludes the CL1 degradation sequence of SEQ ID NO: 71.

In some embodiments of any of the compositions described herein, thevector is 19OTOF48, shown in FIG. 43 .

In some embodiments of any of the compositions described herein, thevector is 1OTOF20.CL1, shown in FIG. 44 . The intron 21 splice donorsequence in 1OTOF20,CL1 is SEQ ID NO: 72.

In some embodiments of any of the compositions described herein, thevector is 21OTOF48.WPRE, shown in FIG. 45 . The intron 21 spliceacceptor sequence is SEQ ID NO: 73.

In some embodiments of any of the compositions described herein, thevector is 1OTOF21.CL1, shown in FIG. 46 . The intron 22 splice donorsequence is SEQ ID NO: 74.

In some embodiments of any of the compositions described herein, thevector is 22OTOF48.WPRE, shown in FIG. 47 . The intron 22 spliceacceptor sequence is SEQ ID NO: 75.

In some embodiments of any of the compositions described herein, thevector is 105.pA.NTF3.CMVd, shown in FIG. 48 . The 105.pA.NTF3.CMVdvector includes the following sequences: SV40 polyA (SEQ ID NO: 76),HSV-TK poly(A) (SEQ ID NO: 77), sequence encoding human NTF3 (SEQ ID NO:79), and CMVd (SEQ ID NO: 86).

In some embodiments, a vector can include a CMV enhancer and a chickenJ-actin promoter, e.g., a sequence of SEQ ID NO: 61.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-eGFP-P2A-5′mOTOF.SD (SEQ ID NO: 87), or is a vectorthat includes a sequence that is at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQID NO: 87. The pAAV-HBA-eGFP-P2A-5′mOTOF.SD vector is 4,472 bp in lengthand is shown in FIG. 53 . The pAAV-HBA-eGFP-P2A-5′mOTOF.SD vectorincludes an AAV ITR (SEQ ID NO: 59), a CMV enhancer (SEQ ID NO: 70), asequence encoding a portion of 5′mOTOF (SEQ ID NO: 94), and a splicedonor sequence (SEQ ID NO: 64).

In some embodiments of any of the compositions described herein, thevector is pAAV-SA-3′mOTOF.WPRE (SEQ ID NO: 88), or is a vector thatincludes a sequence that is at least 80%, at least 82%, at least 84%, atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:88. The pAAV-SA-3′mOTOF.WPRE vector is 4,674 bp in length and is shownin FIG. 54 . The pAAV-SA-3′mOTOF.WPRE vector includes an AAV ITR (SEQ IDNO: 59), a splice acceptor sequence (SEQ ID NO: 65), a sequence encodinga portion 3′mOTOF (SEQ ID NO: 95), a WPRE sequence (SEQ ID NO: 69), aBGHpA sequence (SEQ ID NO: 68), and an AAV ITR sequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-eGFP-P2A-5′mOTOF.SD-AK, shown in FIG. 55 , and is4,549 bp in length. The pAAV-HBA-eGFP-P2A-5′mOTOF.SD-AK vector includesan AAV ITR (SEQ ID NO: 59), a CMV enhancer (SEQ ID NO: 70), a sequenceencoding a portion 5′mOTOF (SEQ ID NO: 94), a SD-intron sequence (SEQ IDNO: 72), an AK sequence (SEQ ID NO: 67), and an AAV ITR (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-AK-SA-3′mOTOF-WPRE, shown in FIG. 56 and is 4,751 bp inlength. The pAAV-AK-SA-3′mOTOF-WPRE vector includes an AAV ITR (SEQ IDNO: 59), an AK sequence (SEQ ID NO: 66), a sequence encoding a portionof 3′ mOTOF (SEQ ID NO: 95), a WPRE sequence (SEQ ID NO: 69), a BGHpAsequence (SEQ ID NO: 68), and an AAV ITR (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-CMV-5′hOTOF-SD-AK (p108 plasmid), shown in FIG. 57 and is4,567 bp in length. The pAAV-CMV-5′hOTOF-SD-AK vector includes an AAVITR (SEQ ID NO: 59), a FVII stuffer (SEQ ID NO: 90), a FVII stuffer (SEQID NO: 91), a CMV enhancer and promoter (SEQ ID NO: 70), a portion of asequence encoding 5′hOTOF (SEQ ID NO: 62), a SD intron sequence (SEQ IDNO: 72), an AK sequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQ IDNO: 60). In some embodiments, the vector that includes a sequence thatis at least 80%, at least 82%, at least 84%, at least 86%, at least 88%,at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, atleast 99%, or 100% identical to SEQ ID NO: 90. In some embodiments, thevector that includes a sequence that is at least 80%, at least 82%, atleast 84%, at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 98%, at least 99%, or 100% identicalto SEQ ID NO: 91.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-5′hOTOF-SD-AP, shown in FIG. 58 , and is 4,540 bp inlength. The pAAV-HBA-5′hOTOF-SD-AP includes an AAV ITR sequence (SEQ IDNO: 59), a CMV enhancer (SEQ ID NO: 70), a sequence encoding a portion5′hOTOF (SEQ ID NO: 62), an AP rec sequence (SEQ ID NO: 89), and an AAVITR sequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-5′hOTOF-SD-AK, shown in FIG. 59 , and is 4,745 bp inlength. The pAAV-HBA-5′hOTOF-SD-AK includes an AAV ITR sequence (SEQ IDNO: 59), a FVIII stuffer (4677-5173) sequence (SEQ ID NO: 90), a FVIIIstuffer (3679-4177) sequence (SEQ ID: 91), a CMV enhancer (SEQ ID NO:70), a sequence encoding a portion 5′hOTOF (SEQ ID NO: 62), and an AAVITR sequence (SEQ ID NO: 60). In some embodiments, the vector includes asequence that is at least 80%, at least 82%, at least 84%, at least 86%,at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 90, or asequence that is at least 80%, at least 82%, at least 84%, at least 86%,at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO: 91.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-5′hOTOFcodop-SD-AK, shown in FIG. 60 , and is 4,745bp in length, The pAAV-CMV-5′hOTOF-SD-AK vector includes an AAV ITRsequence (SEQ ID NO: 59), a FVIII stuffer (4677-5173) sequence (SEQ IDNO: 90), a FVIII stuffer (3679-4177) sequence (SEQ ID: 91), a CMVenhancer (SEQ ID NO: 70), a sequence encoding a portion a 5′OTOF codopsequence (SEQ ID NO: 92), an AK sequence (SEQ ID NO: 67), and an AAV ITRsequence (SEQ ID NO: 60). In some embodiments, the vector that includesa sequence that is at least 80%, at least 82%, at least 84%, at least86%, at least 88%, at least 90%, at least 92%, at least 94%, at least96%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 90. Insome embodiments, the vector that includes a sequence that is at least80%, at least 82%, at least 84%, at least 86%, at least 88%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99%, or 100% identical to SEQ ID NO: 91.

In some embodiments of any of the compositions described herein, thevector is pAAV-HBA-5′hOTOFcodop-SD, shown in FIG. 61 , and is 4,668 bpin length. The pAAV-HBA-5′hOTOFcodop-SD vector includes an AAV ITRsequence (SEQ ID NO: 59), a FVIII stuffer (4677-5173) sequence (SEQ IDNO: 90), a FVIII stuffer (3679-4177) sequence (SEQ ID: 91), a CMVenhancer (SEQ ID NO: 70), a sequence encoding a portion 5′OTOF codopsequence (SEQ ID NO: 92), and an AAV ITR sequence (SEQ ID NO: 60). Insome embodiments, the vector that includes a sequence that is at least80%, at least 82%, at least 84%, at least 86%, at least 88%, at least90%, at least 92%, at least 94%, at least 96%, at least 98%, at least99%, or 100% identical to SEQ ID NO: 90. In some embodiments, the vectorthat includes a sequence that is at least 80%, at least 82%, at least84%, at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 98%, at least 99%, or 100% identical to SEQID NO: 91.

In some embodiments of any of the compositions described herein, thevector is pAAV-CMV-5′hOTOF-SD, shown in FIG. 62 , and is 4,490 bp inlength. The pAAV-CMV-5′hOTOF-SD vector includes an AAV ITR sequence (SEQID NO: 59), two FVIII stuffer sequences, a CMV enhancer and promoter(SEQ ID NO: 70), a sequence encoding a portion of a 5′OTOF codopsequence (SEQ ID NO: 92), a SD-intron sequence (SEQ ID NO: 64), and anAAV ITR sequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-CMV-5′hOTOF-SD-AK, shown in FIG. 63 , and is 4,567 bp inlength. The pAAV-CMV-5′hOTOF-SD-AK vector includes an AAV ITR sequence(SEQ ID NO: 59), two FVIII stuffer sequences, a CMV enhancer andpromoter (SEQ ID NO: 70), a sequence encoding a portion of a 5′OTOFcodop sequence (SEQ ID NO: 92), a splice donor (SD) intron sequence (SEQID NO: 64), an AK sequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-CBA-5′hOTOF-SD-AK, shown in FIG. 64 , and is 4,711 bp inlength. The pAAV-CBA-5′hOTOF-SD-AK vector includes an AAV ITR sequence(SEQ ID NO: 59), a CMV enhancer and chicken beta-actin promoter (SEQ IDNO: 61), a chimeric intronic sequence, a sequence encoding a portion ofa 5′hOTOF codop sequence (SEQ ID NO: 92), an intronic sequence, an AKsequence (SEQ ID NO: 67), and an AAV ITR sequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-CBA-5′hOTOF-SD, shown in FIG. 65 , and is 4,634 bp inlength. The pAAV-CBA-5′hOTOF-SD vector includes an AAV ITR sequence (SEQID NO: 59), a CMV enhancer and beta-actin promoter (SEQ ID NO: 61), achimeric intronic sequence, a sequence encoding a portion of a 5′OTOFcodop sequence (SEQ ID NO: 92), an intronic sequence, and an AAV ITRsequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-SA-3′OTOF, shown in FIG. 66 , and is 4,587 bp in length.The pAAV-CBA-5′hOTOF-SD vector includes an AAV ITR sequence (SEQ ID NO:59), a splice acceptor sequence (SEQ ID NO: 65), a sequence encoding aportion 3′OTOF sequence (SEQ ID NO: 63), a bGHpA sequence (SEQ ID NO:68), a FVIII stuffer 9 sequence (SEQ ID NO: 57), and an AAV ITR sequence(SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-AP-SA-3′OTOF, shown in FIG. 67 , and is 4,959 bp inlength. The pAAV-AP-SA-3′OTOF vector includes an AAV ITR sequence (SEQID NO: 59), an AP rec sequence (SEQ ID NO: 89), a splice acceptorsequence (SEQ ID NO: 65), a sequence encoding a portion of a 3′OTOFsequence (SEQ ID NO: 63), a bGHpolyA sequence (SEQ ID NO: 68), and anAAV ITR sequence (SEQ ID NO: 60).

In some embodiments of any of the compositions described herein, thevector is pAAV-AK-SA-3′OTOFcodop, shown in FIG. 68 , and is 4,664 bp inlength. The pAAV-AK-SA-3′OTOFcodop vector includes an AAV ITR (SEQ IDNO: 59), an AK sequence (SEQ ID NO: 66), a sequence encoding a portionof a 3′OTOF codop sequence (SEQ ID NO: 93), a bGH polyA sequence (SEQ IDNO: 68), and an AAV ITR sequence (SEQ ID NO: 60).

The vectors provided herein can be of different sizes. The choice ofvector that is used in any of the compositions, kits, and methodsdescribed herein may depend on the size of the vector.

In some embodiments, the vector(s) is a plasmid and can include a totallength of up to about 1 kb, up to about 2 kb, up to about 3 kb, up toabout 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up toabout 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, upto about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15kb. In some embodiments, the vector(s) is a plasmid and can have a totallength in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb,about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb toabout 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14kb, about 1 kb to about 15 kb, about 2 kb to about 3 kb, about 2 kb toabout 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb,about 2 kb to about 10 kb, about 2 kb to about 11 kb, about 2 kb toabout 12 kb, about 2 kb to about 13 kb, about 2 kb to about 14 kb, about2 kb to about 15 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb,about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 3 kb toabout 11 kb, about 3 kb to about 12 kb, about 3 kb to about 13 kb, about3 kb to about 14 kb, about 3 kb to about 15 kb, about 4 kb to about 5kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb toabout 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 4kb to about 11 kb, about 4 kb to about 12 kb, about 4 kb to about 13 kb,about 4 kb to about 14 kb, about 4 kb to about 15 kb, about 5 kb toabout 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5kb to about 9 kb, about 5 kb to about 10 kb, about 5 kb to about 11 kb,about 5 kb to about 12 kb, about 5 kb to about 13 kb, about 5 kb toabout 14 kb, or about 5 kb to about 15 kb.

In some embodiments, the vector(s) is a transposon (e.g., PiggyBactransposon) and can include greater than 200 kb. In some examples, thevector(s) is a transposon having a total length in the range of about 1kb to about 10 kb, about 1 kb to about 20 kb, about 1 kb to about 30 kb,about 1 kb to about 40 kb, about 1 kb to about 50 kb, about 1 kb toabout 60 kb, about 1 kb to about 70 kb, about 1 kb to about 80 kb, about1 kb to about 90 kb, about 10 kb to about 20 kb, about 10 kb to about 30kb, about 10 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kbto about 60 kb, about 10 kb to about 70 kb, about 10 kb to about 90 kb,about 10 kb to about 100 kb, about 20 kb to about 30 kb, about 20 kb toabout 40 kb, about 20 kb to about 50 kb, about 20 kb to about 60 kb,about 20 kb to about 70 kb, about 20 kb to about 80 kb, about 20 kb toabout 90 kb, about 20 kb to about 100 kb, about 30 kb to about 40 kb,about 30 kb to about 50 kb, about 30 kb to about 60 kb, about 30 kb toabout 70 kb, about 30 kb to about 80 kb, about 30 kb to about 90 kb,about 30 kb to about 100 kb, about 40 kb to about 50 kb, about 40 kb toabout 60 kb, about 40 kb to about 70 kb, about 40 kb to about 80 kb,about 40 kb to about 90 kb, about 40 kb to about 100 kb, about 50 kb toabout 60 kb, about 50 kb to about 70 kb, about 50 kb to about 80 kb,about 50 kb to about 90 kb, about 50 kb to about 100 kb, about 60 kb toabout 70 kb, about 60 kb to about 80 kb, about 60 kb to about 90 kb,about 60 kb to about 100 kb, about 70 kb to about 80 kb, about 70 kb toabout 90 kb, about 70 kb to about 100 kb, about 80 kb to about 90 kb,about 80 kb to about 100 kb, about 90 kb to about 100 kb, about 1 kb toabout 100 kb, about 100 kb to about 200 kb, about 100 kb to about 300kb, about 100 kb to about 400 kb, or about 100 kb to about 500 kb.

In some embodiments, the vector is a cosmid and can have a total lengthof up to 55 kb. In some examples, the vector is a cosmid and has a totalnumber of nucleotides of about 1 kb to about 10 kb, about 1 kb to about20 kb, about 1 kb to about 30 kb, about 1 kb to about 40 kb, about 1 kbto about 50 kb, about 1 kb to about 55 kb, about 10 kb to about 20 kb,about 10 kb to about 30 kb, about 10 kb to about 40 kb, about 10 kb toabout 50 kb, about 10 kb to about 55 kb, about 15 kb to about 55 kb,about 15 kb to about 50 kb, about 15 kb to about 40 kb, about 15 kb toabout 30 kb, about 15 kb to about 20 kb, about 20 kb to about 55 kb,about 20 kb to about 50 kb, about 20 kb to about 40 kb, about 20 kb toabout 30 kb, about 25 kb to about 55 kb, about 25 kb to about 50 kb,about 25 kb to about 40 kb, about 25 kb to about 30 kb, about 30 kb toabout 55 kb, about 30 kb to about 50 kb, about 30 kb to about 40 kb,about 35 kb to about 55 kb, about 40 kb to about 55 kb, about 40 kb toabout 50 kb, or about 45 kb to about 55 kb.

In some embodiments, the vector(s) is an artificial chromosome and canhave a total number of nucleotides of about 100 kb to about 2000 kb. Insome embodiments, the artificial chromosome(s) is a human artificialchromosome (HAC) and can have a total number of nucleotides in the rangeof about 1 kb to about 10 kb, 1 kb to about 20 kb, about 1 kb to about30 kb, about 1 kb to about 40 kb, about 1 kb to about 50 kb, about 1 kbto about 60 kb, about 10 kb to about 20 kb, about 10 kb to about 30 kb,about 10 kb to about 40 kb, about 10 kb to about 50 kb, about 10 kb toabout 60 kb, about 20 kb to about 30 kb, about 20 kb to about 40 kb,about 20 kb to about 50 kb, about 20 kb to about 60 kb, about 30 kb toabout 40 kb, about 30 kb to about 50 kb, about 30 kb to about 60 kb,about 40 kb to about 50 kb, about 40 kb to about 60 kb, or about 50 kbto about 60 kb.

In some embodiments, the artificial chromosome(s) is a yeast artificialchromosome (YAC) and can have a total number of nucleotides up to 1000kb. In some embodiments, the artificial chromosome(s) is a YAC having atotal number of nucleotides in the range of about 100 kb to about 1,000kb, about 100 kb to about 900 kb, about 100 kb to about 800 kb, about100 kb to about 700 kb, about 100 kb to about 600 kb, about 100 kb toabout 500 kb, about 100 kb to about 400 kb, about 100 kb to about 300kb, about 100 kb to about 200 kb, about 200 kb to about 1,000 kb, about200 kb to about 900 kb, about 200 kb to about 800 kb, about 200 kb toabout 700 kb, about 200 kb to about 600 kb, about 200 kb to about 500kb, about 200 kb to about 400 kb, about 200 kb to about 300 kb, about300 kb to about 1,000 kb, about 300 kb to about 900 kb, about 300 kb toabout 800 kb, about 300 kb to about 700 kb, about 300 kb to about 600kb, about 300 kb to about 500 kb, about 300 kb to about 400 kb, about400 kb to about 1,000 kb, about 400 kb to about 900 kb, about 400 kb toabout 800 kb, about 400 kb to about 700 kb, about 400 kb to about 600kb, about 400 kb to about 500 kb, about 500 kb to about 1,000 kb, about500 kb to about 900 kb, about 500 kb to about 800 kb, about 500 kb toabout 700 kb, about 500 kb to about 600 kb, about 600 kb to about 1,000kb, about 600 kb to about 900 kb, about 600 kb to about 800 kb, about600 kb to about 700 kb, about 700 kb to about 1,000 kb, about 700 kb toabout 900 kb, about 700 kb to about 800 kb, about 800 kb to about 1,000kb, about 800 kb to about 900 kb, or about 900 kb to about 1,000 kb.

In some embodiments, the artificial chromosome(s) is a bacterialartificial chromosome (BAC) and can have a total number of nucleotidesof up to 750 kb. In some embodiments, the artificial chromosome(s) is aBAC and can have a total number of nucleotides in the range of about 100kb to about 750 kb, about 100 kb to about 700 kb, about 100 kb to about600 kb, about 100 kb to about 500 kb, about 100 kb to about 400 kb,about 100 kb to about 300 kb, about 100 kb to about 200 kb, about 150 kbto about 750 kb, about 150 kb to about 700 kb, about 150 kb to about 600kb, about 150 kb to about 500 kb, about 150 kb to about 400 kb, about150 kb to about 300 kb, about 150 kb to about 200 kb, about 200 kb toabout 750 kb, about 200 kb to about 700 kb, about 200 kb to about 600kb, about 200 kb to about 500 kb, about 200 kb to about 400 kb, about200 kb to about 300 kb, about 250 kb to about 750 kb, about 250 kb toabout 700 kb, about 250 kb to about 600 kb, about 250 kb to about 500kb, about 250 kb to about 400 kb, about 250 kb to about 300 kb, about300 kb to about 750 kb, about 300 kb to about 700 kb, about 300 kb toabout 600 kb, about 300 kb to about 500 kb, about 300 kb to about 400kb, about 350 kb to about 750 kb, about 350 kb to about 700 kb, about350 kb to about 600 kb, about 350 kb to about 500 kb, about 350 kb toabout 400 kb, about 400 kb to about 750 kb, about 400 kb to about 700kb, about 450 kb to about 600 kb, about 450 kb to about 500 kb, about500 kb to about 750 kb, about 500 kb to about 700 kb, about 500 kb toabout 600 kb, about 550 kb to about 750 kb, about 550 kb to about 700kb, about 550 kb to about 600 kb, about 600 kb to about 750 kb, about600 kb to about 700 kb, or about 650 kb to about 750 kb.

In some embodiments, the artificial chromosome(s) is a P1-derivedartificial chromosome (PAC) and can have a total number of nucleotidesof up to 300 kb. In some embodiments, the P1-derived artificialchromosome(s) can have a total number of nucleotides in the range ofabout 100 kb to about 300 kb, about 100 kb to about 200 kb, or about 200kb to about 300 kb.

In some embodiments, the vector(s) is a viral vector and can have atotal number of nucleotides of up to 10 kb. In some embodiments, theviral vector(s) can have a total number of nucleotides in the range ofabout 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb,about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb toabout 10 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb,about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb toabout 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb,about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb toabout 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb,about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb toabout 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, orabout 9 kb to about 10 kb.

In some embodiments, the vector(s) is a lentivirus and can have a totalnumber of nucleotides of up to 8 kb. In some examples, thelentivirus(es) can have a total number of nucleotides of about 1 kb toabout 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb,about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb toabout 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb,about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb toabout 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

In some embodiments, the vector(s) is an adenovirus and can have a totalnumber of nucleotides of up to 8 kb. In some embodiments, theadenovirus(es) can have a total number of nucleotides in the range ofabout 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb toabout 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb,about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb toabout 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb,about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kbto about 8 kb.

In some embodiments, the vector(s) is an adeno-associated virus (AAVvector) and can include a total number of nucleotides of up to 5 kb. Insome embodiments, the AAV vector(s) can include a total number ofnucleotides in the range of about 1 kb to about 2 kb, about 1 kb toabout 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 2kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb,about 3 kb to about 4 kb, about 3 kb to about 5 kb, or about 4 kb toabout 5 kb.

In some embodiments, the vector(s) is a Gateway® vector and can includea total number of nucleotides of up to 5 kb. In some embodiments, eachGateway® vector(s) includes a total number of nucleotides in the rangeof about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb toabout 4 kb, about 1 kb to about 5 kb, about 2 kb to about 3 kb, about 2kb to about 4 kb, about 2 kb to about 5 kb, about 3 kb to about 4 kb,about 3 kb to about 5 kb, or about 4 kb to about 5 kb.

In some embodiments of any of the compositions, kits, and methodsprovided herein, the at least two different vectors can be substantiallythe same type of vector and may differ in size. In some embodiments, theat least two different vectors can be different types of vector, and mayhave substantially the same size or have different sizes.

In some embodiments, any of the at least two vectors can have a totalnumber of nucleotides in the range of about 500 nucleotides to about10,000 nucleotides, about 500 nucleotides to about 9,500 nucleotides,about 500 nucleotides to about 9,000 nucleotides, about 500 nucleotidesto about 8,500 nucleotides, about 500 nucleotides to about 8,000nucleotides, about 500 nucleotides to about 7,800 nucleotides, about 500nucleotides to about 7,600 nucleotides, about 500 nucleotides to about7,400 nucleotides, about 500 nucleotides to about 7,200 nucleotides,about 500 nucleotides to about 7,000 nucleotides, about 500 nucleotidesto about 6,800 nucleotides, about 500 nucleotides to about 6,600nucleotides, about 500 nucleotides to about 6,400 nucleotides, about 500nucleotides to about 6,200 nucleotides, about 500 nucleotides to about6,000 nucleotides, about 500 nucleotides to about 5,800 nucleotides,about 500 nucleotides to about 5,600 nucleotides, about 500 nucleotidesto about 5,400 nucleotides, about 500 nucleotides to about 5,200nucleotides, about 500 nucleotides to about 5,000 nucleotides, about 500nucleotides to about 4,800 nucleotides, about 4,600 nucleotides, about500 nucleotides to about 4,400 nucleotides, about 500 nucleotides toabout 4,200 nucleotides, about 500 nucleotides to about 4,000nucleotides, about 500 nucleotides to about 3,800 nucleotides, about 500nucleotides to about 3,600 nucleotides, about 500 nucleotides to about3,400 nucleotides, about 500 nucleotides to about 3,200 nucleotides,about 500 nucleotides to about 3,000 nucleotides, about 500 nucleotidesto about 2,800 nucleotides, about 500 nucleotides to about 2,600nucleotides, about 500 nucleotides to about 2,400 nucleotides, about 500nucleotides to about 2,200 nucleotides, about 500 nucleotides to about2,000 nucleotides, about 500 nucleotides to about 1,800 nucleotides,about 500 nucleotides to about 1,600 nucleotides, about 500 nucleotidesto about 1,400 nucleotides, about 500 nucleotides to about 1,200nucleotides, about 500 nucleotides to about 1,000 nucleotides, about 500nucleotides to about 800 nucleotides, about 800 nucleotides to about10,000 nucleotides, about 800 nucleotides to about 9,500 nucleotides,about 800 nucleotides to about 9,000 nucleotides, about 800 nucleotidesto about 8,500 nucleotides, about 800 nucleotides to about 8,000nucleotides, about 800 nucleotides to about 7,800 nucleotides, about 800nucleotides to about 7,600 nucleotides, about 800 nucleotides to about7,400 nucleotides, about 800 nucleotides to about 7,200 nucleotides,about 800 nucleotides to about 7,000 nucleotides, about 800 nucleotidesto about 6,800 nucleotides, about 800 nucleotides to about 6,600nucleotides, about 800 nucleotides to about 6,400 nucleotides, about 800nucleotides to about 6,200 nucleotides, about 800 nucleotides to about6,000 nucleotides, about 800 nucleotides to about 5,800 nucleotides,about 800 nucleotides to about 5,600 nucleotides, about 800 nucleotidesto about 5,400 nucleotides, about 800 nucleotides to about 5,200nucleotides, about 800 nucleotides to about 5,000 nucleotides, about 800nucleotides to about 4,800 nucleotides, about 800 nucleotides to about4,600 nucleotides, about 800 nucleotides to about 4,400 nucleotides,about 800 nucleotides to about 4,200 nucleotides, about 800 nucleotidesto about 4,000 nucleotides, about 800 nucleotides to about 3,800nucleotides, about 800 nucleotides to about 3,600 nucleotides, about 800nucleotides to about 3,400 nucleotides, about 800 nucleotides to about3,200 nucleotides, about 800 nucleotides to about 3,000 nucleotides,about 800 nucleotides to about 2,800 nucleotides, about 800 nucleotidesto about 2,600 nucleotides, about 800 nucleotides to about 2,400nucleotides, about 800 nucleotides to about 2,200 nucleotides, about 800nucleotides to about 2,000 nucleotides, about 800 nucleotides to about1,800 nucleotides, about 800 nucleotides to about 1,600 nucleotides,about 800 nucleotides to about 1,400 nucleotides, about 800 nucleotidesto about 1,200 nucleotides, about 800 nucleotides to about 1,000nucleotides, about 1,000 nucleotides to about 10,000 nucleotides, about1,000 nucleotides to about 9,000 nucleotides, about 1,000 nucleotides toabout 8,500 nucleotides, about 1,000 nucleotides to about 8,000nucleotides, about 1,000 nucleotides to about 7,800 nucleotides, about1,000 nucleotides to about 7,600 nucleotides, about 1,000 nucleotides toabout 7,400 nucleotides, about 1,000 nucleotides to about 7,200nucleotides, about 1,000 nucleotides to about 7,000 nucleotides, about1,000 nucleotides to about 6,800 nucleotides, about 1,000 nucleotides toabout 6,600 nucleotides, about 1,000 nucleotides to about 6,400nucleotides, about 1,000 nucleotides to about 6,200 nucleotides, about1,000 nucleotides to about 6,000 nucleotides, about 1,000 nucleotides toabout 5,800 nucleotides, about 1,000 nucleotides to about 5,600nucleotides, about 1,000 nucleotides to about 5,400 nucleotides, about1,000 nucleotides to about 5,200 nucleotides, about 1,000 nucleotides toabout 5,000 nucleotides, about 1,000 nucleotides to about 4,800nucleotides, about 1,000 nucleotides to about 4,600 nucleotides, about1,000 nucleotides to about 4,400 nucleotides, about 1,000 nucleotides toabout 4,200 nucleotides, about 1,000 nucleotides to about 4,000nucleotides, about 1,000 nucleotides to about 3,800 nucleotides, about1,000 nucleotides to about 3,600 nucleotides, about 1,000 nucleotides toabout 3,400 nucleotides, about 1,000 nucleotides to about 3,200nucleotides, about 1,000 nucleotides to about 3,000 nucleotides, about1,000 nucleotides to about 2,600 nucleotides, about 1,000 nucleotides toabout 2,400 nucleotides, about 1,000 nucleotides to about 2,200nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about1,000 nucleotides to about 1,800 nucleotides, about 1,000 nucleotides toabout 1,600 nucleotides, about 1,000 nucleotides to about 1,400nucleotides, about 1,000 nucleotides to about 1,200 nucleotides, about1,200 nucleotides to about 10,000 nucleotides, about 1,200 nucleotidesto about 9,500 nucleotides, about 1,200 nucleotides to about 9,000nucleotides, about 1,200 nucleotides to about 8,500 nucleotides, about1,200 nucleotides to about 8,000 nucleotides, about 1,200 nucleotides toabout 7,800 nucleotides, about 1,200 nucleotides to about 7,600nucleotides, about 1,200 nucleotides to about 7,400 nucleotides, about1,200 nucleotides to about 7,200 nucleotides, about 1,200 nucleotides toabout 7,000 nucleotides, about 1,200 nucleotides to about 6,800nucleotides, about 1,200 nucleotides to about 6,600 nucleotides, about1,200 nucleotides to about 6,400 nucleotides, about 1,200 nucleotides toabout 6,200 nucleotides, about 1,200 nucleotides to about 6,000nucleotides, about 1,200 nucleotides to about 5,800 nucleotides, about1,200 nucleotides to about 5,600 nucleotides, about 1,200 nucleotides toabout 5,400 nucleotides, about 1,200 nucleotides to about 5,000nucleotides, about 1,200 nucleotides to about 4,800 nucleotides, about1,200 nucleotides to about 4,600 nucleotides, about 1,200 nucleotides toabout 4,400 nucleotides, about 1,200 nucleotides to about 4,200nucleotides, about 1,200 nucleotides to about 4,000 nucleotides, about1,200 nucleotides to about 3,800 nucleotides, about 1,200 nucleotides toabout 3,600 nucleotides, about 1,200 nucleotides to about 3,400nucleotides, about 1,200 nucleotides to about 3,200 nucleotides, about1,200 nucleotides to about 3,000 nucleotides, about 1,200 nucleotides toabout 2,800 nucleotides, about 1,200 nucleotides to about 2,600nucleotides, about 1,200 nucleotides to about 2,400 nucleotides, about1,200 nucleotides to about 2,200 nucleotides, about 1,200 nucleotides toabout 2,000 nucleotides, about 1,200 nucleotides to about 1,800nucleotides, about 1,200 nucleotides to about 1,600 nucleotides, about1,200 nucleotides to about 1,400 nucleotides, about 1,400 nucleotides toabout 10,000 nucleotides, about 1,400 nucleotides to about 9,500nucleotides, about 1,400 nucleotides to about 9,000 nucleotides, about1,400 nucleotides to about 8,500 nucleotides, about 1,400 nucleotides toabout 8,000 nucleotides, about 1,400 nucleotides to about 7,800nucleotides, about 1,400 nucleotides to about 7,600 nucleotides, about1,400 nucleotides to about 7,400 nucleotides, about 1,400 nucleotides toabout 7,200 nucleotides, about 1,400 nucleotides to about 7,000nucleotides, about 1,400 nucleotides to about 6,800 nucleotides, about1,400 nucleotides to about 6,600 nucleotides, about 1,400 nucleotides toabout 6,400 nucleotides, about 1,400 nucleotides to about 6,200nucleotides, about 1,400 nucleotides to about 6,000 nucleotides, about1,400 nucleotides to about 5,800 nucleotides, about 1,400 nucleotides toabout 5,600 nucleotides, about 1,400 nucleotides to about 5,400nucleotides, about 1,400 nucleotides to about 5,200 nucleotides, about1,400 nucleotides to about 5,000 nucleotides, about 1,400 nucleotides toabout 4,800 nucleotides, about 1,400 nucleotides to about 4,600nucleotides, about 1,400 nucleotides to about 4,400 nucleotides, about1,400 nucleotides to about 4,200 nucleotides, about 1,400 nucleotides toabout 4,000 nucleotides, about 1,400 nucleotides to about 3,800nucleotides, about 1,400 nucleotides to about 3,600 nucleotides, about1,400 nucleotides to about 3,400 nucleotides, about 1,400 nucleotides toabout 3,200 nucleotides, about 1,400 nucleotides to about 3,000nucleotides, about 1,400 nucleotides to about 2,600 nucleotides, about1,400 nucleotides to about 2,400 nucleotides, about 1,400 nucleotides toabout 2,200 nucleotides, about 1,400 nucleotides to about 2,000nucleotides, about 1,400 nucleotides to about 1,800 nucleotides, about1,400 nucleotides to about 1,600 nucleotides, about 1,600 nucleotides toabout 10,000 nucleotides, about 1,600 nucleotides to about 9,500nucleotides, about 1,600 nucleotides to about 9,000 nucleotides, about1,600 nucleotides to about 8,500 nucleotides, about 1,600 nucleotides toabout 8,000 nucleotides, about 1,600 nucleotides to about 7,800nucleotides, about 1,600 nucleotides to about 7,600 nucleotides, about1,600 nucleotides to about 7,400 nucleotides, about 1,600 nucleotides toabout 7,200 nucleotides, about 1,600 nucleotides to about 7,000nucleotides, about 1,600 nucleotides to about 6,800 nucleotides, about1,600 nucleotides to about 6,400 nucleotides, about 1,600 nucleotides toabout 6,200 nucleotides, about 1,600 nucleotides to about 6,000nucleotides, about 1,600 nucleotides to about 5,800 nucleotides, about1,600 nucleotides to about 5,600 nucleotides, about 1,600 nucleotides toabout 5,400 nucleotides, about 1,600 nucleotides to about 5,200nucleotides, about 1,600 nucleotides to about 5,000 nucleotides, about1,600 nucleotides to about 4,800 nucleotides, about 1,600 nucleotides toabout 4,600 nucleotides, about 1,600 nucleotides to about 4,400nucleotides, about 1,600 nucleotides to about 4,200 nucleotides, about1,600 nucleotides to about 4,000 nucleotides, about 1,600 nucleotides toabout 3,800 nucleotides, about 1,600 nucleotides to about 3,600nucleotides, about 1,600 nucleotides to about 3,400 nucleotides, about1,600 nucleotides to about 3,200 nucleotides, about 1,600 nucleotides toabout 3,000 nucleotides, about 1,600 nucleotides to about 2,800nucleotides, about 1,600 nucleotides to about 2,600 nucleotides, about1,600 nucleotides to about 2,400 nucleotides, about 1,600 nucleotides toabout 2,200 nucleotides, about 1,600 nucleotides to about 2,000nucleotides, about 1,600 nucleotides to about 1,800 nucleotides, about1,800 nucleotides to about 10,000 nucleotides, about 1,800 nucleotidesto about 9,500 nucleotides, about 1,800 nucleotides to about 9,000nucleotides, about 1,800 nucleotides to about 8,500 nucleotides, about1,800 nucleotides to about 8,000 nucleotides, about 1,800 nucleotides toabout 7,800 nucleotides, about 1,800 nucleotides to about 7,600nucleotides, about 1,800 nucleotides to about 7,400 nucleotides, about1,800 nucleotides to about 7,200 nucleotides, about 1,800 nucleotides toabout 7,000 nucleotides, about 1,800 nucleotides to about 6,800nucleotides, about 1,800 nucleotides to about 6,600 nucleotides, about1,800 nucleotides to about 6,400 nucleotides, about 1,800 nucleotides toabout 6,200 nucleotides, about 1,800 nucleotides to about 6,000nucleotides, about 1,800 nucleotides to about 5,800 nucleotides, about1,800 nucleotides to about 5,600 nucleotides, about 1,800 nucleotides toabout 5,400 nucleotides, about 1,800 nucleotides to about 5,200nucleotides, about 1,800 nucleotides to about 5,000 nucleotides, about1,800 nucleotides to about 4,800 nucleotides, about 1,800 nucleotides toabout 4,600 nucleotides, about 1,800 nucleotides to about 4,400nucleotides, about 1,800 nucleotides to about 4,200 nucleotides, about1,800 nucleotides to about 4,000 nucleotides, about 1,800 nucleotides toabout 3,800 nucleotides, about 1,800 nucleotides to about 3,600nucleotides, about 1,800 nucleotides to about 3,400 nucleotides, about1,800 nucleotides to about 3,200 nucleotides, about 1,800 nucleotides toabout 3,000 nucleotides, about 1,800 nucleotides to about 2,800nucleotides, about 1,800 nucleotides to about 2,600 nucleotides, about1,800 nucleotides to about 2,400 nucleotides, about 1,800 nucleotides toabout 2,200 nucleotides, about 1,800 nucleotides to about 2,000nucleotides, about 2,000 nucleotides to about 10,000 nucleotides, about2,000 nucleotides to about 9,500 nucleotides, about 2,000 nucleotides toabout 9,000 nucleotides, about 2,000 nucleotides to about 8,500nucleotides, about 2,000 nucleotides to about 8,000 nucleotides, about2,000 nucleotides to about 7,800 nucleotides, about 2,000 nucleotides toabout 7,600 nucleotides, about 2,000 nucleotides to about 7,400nucleotides, about 2,000 nucleotides to about 7,200 nucleotides, about2,000 nucleotides to about 7,000 nucleotides, about 2,000 nucleotides toabout 6,800 nucleotides, about 2,000 nucleotides to about 6,600nucleotides, about 2,000 nucleotides to about 6,400 nucleotides, about2,000 nucleotides to about 6,200 nucleotides, about 2,000 nucleotides toabout 6,000 nucleotides, about 2,000 nucleotides to about 5,800nucleotides, about 2,000 nucleotides to about 5,600 nucleotides, about2,000 nucleotides to about 5,400 nucleotides, about 2,000 nucleotides toabout 5,200 nucleotides, about 2,000 nucleotides to about 5,000nucleotides, about 2,000 nucleotides to about 4,800 nucleotides, about2,000 nucleotides to about 4,600 nucleotides, about 2,000 nucleotides toabout 4,400 nucleotides, about 2,000 nucleotides to about 4,200nucleotides, about 2,000 nucleotides to about 4,000 nucleotides, about2,000 nucleotides to about 3,800 nucleotides, about 2,000 nucleotides toabout 3,600 nucleotides, about 2,000 nucleotides to about 3,400nucleotides, about 2,000 nucleotides to about 3,200 nucleotides, about2,000 nucleotides to about 3,000 nucleotides, about 2,000 nucleotides toabout 2,800 nucleotides, about 2,000 nucleotides to about 2,600nucleotides, about 2,000 nucleotides to about 2,400 nucleotides, about2,000 nucleotides to about 2,200 nucleotides, about 2,200 nucleotides toabout 10,000 nucleotides, about 9,500 nucleotides, about 9,000nucleotides, about 8,500 nucleotides, about 8,000 nucleotides, about7,800 nucleotides, about 7,600 nucleotides, about 7,400 nucleotides,about 7,200 nucleotides, about 7,000 nucleotides, about 6,800nucleotides, about 6,600 nucleotides, about 6,400 nucleotides, about6,200 nucleotides, about 6,000 nucleotides, about 5,800 nucleotides,about 5,600 nucleotides, about 5,400 nucleotides, about 5,200nucleotides, about 5,000 nucleotides, about 4,800 nucleotides, about4,600 nucleotides, about 4,400 nucleotides, about 4,200 nucleotides,about 4,000 nucleotides, about 3,800 nucleotides, about 3,600nucleotides, about 3,400 nucleotides, about 3,200 nucleotides, about3,000 nucleotides, about 2,800 nucleotides, about 2,600 nucleotides,about 2,400 nucleotides, about 2,400 nucleotides to about 10,000nucleotides, about 2,400 nucleotides to about 9,500 nucleotides, about2,400 nucleotides to about 9,000 nucleotides, about 2,400 nucleotides toabout 8,500 nucleotides, about 2,400 nucleotides to about 8,000nucleotides, about 2,400 nucleotides to about 7,800 nucleotides, about2,400 nucleotides to about 7,600 nucleotides, about 2,400 nucleotides toabout 7,400 nucleotides, about 2,400 nucleotides to about 7,200nucleotides, about 2,400 nucleotides to about 7,000 nucleotides, about2,400 nucleotides to about 6,800 nucleotides, about 2,400 nucleotides toabout 6,600 nucleotides, about 2,400 nucleotides to about 6,400nucleotides, about 2,400 nucleotides to about 6,200 nucleotides, about2,400 nucleotides to about 6,000 nucleotides, about 2,400 nucleotides toabout 5,800 nucleotides, about 2,400 nucleotides to about 5,600nucleotides, about 2,400 nucleotides to about 5,400 nucleotides, about21400 nucleotides to about 5,200 nucleotides, about 2,400 nucleotides toabout 5,000 nucleotides, about 2,400 nucleotides to about 4,800nucleotides, about 2,400 nucleotides to about 4,600 nucleotides, about2,400 nucleotides to about 4,400 nucleotides, about 2,400 nucleotides toabout 4,200 nucleotides, about 2,400 nucleotides to about 4,000nucleotides, about 2,400 nucleotides to about 3,800 nucleotides, about2,400 nucleotides to about 3,600 nucleotides, about 2,400 nucleotides toabout 3,400 nucleotides, about 2,400 nucleotides to about 3,200nucleotides, about 2,400 nucleotides to about 3,000 nucleotides, about2,400 nucleotides to about 2,800 nucleotides, about 2,400 nucleotides toabout 2,600 nucleotides, about 2,600 nucleotides to about 10,000nucleotides, about 2,600 nucleotides to about 9,500 nucleotides, about2,600 nucleotides to about 9,000 nucleotides, about 2,600 nucleotides toabout 8,500 nucleotides, about 2,600 nucleotides to about 8,000nucleotides, about 2,600 nucleotides to about 7,800 nucleotides, about2,600 nucleotides to about 7,600 nucleotides, about 2,600 nucleotides toabout 7,400 nucleotides, about 2,600 nucleotides to about 7,200nucleotides, about 2,600 nucleotides to about 7,000 nucleotides, about2,600 nucleotides to about 6,800 nucleotides, about 2,600 nucleotides toabout 6,600 nucleotides, about 2,600 nucleotides to about 6,400nucleotides, about 2,600 nucleotides to about 6,200 nucleotides, about2,600 nucleotides to about 6,000 nucleotides, about 2,600 nucleotides toabout 5,800 nucleotides, about 2,600 nucleotides to about 5,600nucleotides, about 2,600 nucleotides to about 5,400 nucleotides, about2,600 nucleotides to about 5,200 nucleotides, about 2,600 nucleotides toabout 5,000 nucleotides, about 2,600 nucleotides to about 4,800nucleotides, about 2,600 nucleotides to about 4,600 nucleotides, about2,600 nucleotides to about 4,400 nucleotides, about 2,600 nucleotides toabout 4,200 nucleotides, about 2,600 nucleotides to about 4,000nucleotides, about 2,600 nucleotides to about 3,800 nucleotides, about2,600 nucleotides to about 3,600 nucleotides, about 2,600 nucleotides toabout 3,400 nucleotides, about 2,600 nucleotides to about 3,200nucleotides, about 2,600 nucleotides to about 3,000 nucleotides, about2,600 nucleotides to about 2,800 nucleotides, about 2,800 nucleotides toabout 10,000 nucleotides, about 2,800 nucleotides to about 9,500nucleotides, about 2,800 nucleotides to about 9,000 nucleotides, about2,800 nucleotides to about 8,500 nucleotides, about 2,800 nucleotides toabout 8,000 nucleotides, about 2,800 nucleotides to about 7,800nucleotides, about 2,800 nucleotides to about 7,600 nucleotides, about2,800 nucleotides to about 7,400 nucleotides, about 2,800 nucleotides toabout 7,200 nucleotides, about 2,800 nucleotides to about 7,000nucleotides, about 2,800 nucleotides to about 6,800 nucleotides, about2,800 nucleotides to about 6,600 nucleotides, about 2,800 nucleotides toabout 6,400 nucleotides, about 2,800 nucleotides to about 6,200nucleotides, about 2,800 nucleotides to about 6,000 nucleotides, about2,800 nucleotides to about 5,800 nucleotides, about 2,800 nucleotides toabout 5,600 nucleotides, about 2,800 nucleotides to about 5,400nucleotides, about 2,800 nucleotides to about 5,200 nucleotides, about2,800 nucleotides to about 5,000 nucleotides, about 2,800 nucleotides toabout 4,800 nucleotides, about 2,800 nucleotides to about 4,600nucleotides, about 2,800 nucleotides to about 4,400 nucleotides, about2,800 nucleotides to about 4,200 nucleotides, about 2,800 nucleotides toabout 4,000 nucleotides, about 2,800 nucleotides to about 3,800nucleotides, about 2,800 nucleotides to about 3,600 nucleotides, about2,800 nucleotides to about 3,400 nucleotides, about 2,800 nucleotides toabout 3,200 nucleotides, about 2,800 nucleotides to about 3,000nucleotides, about 3,000 nucleotides to about 10,000 nucleotides, about3,000 nucleotides to about 9,500 nucleotides, about 3,000 nucleotides toabout 9,000 nucleotides, about 3,000 nucleotides to about 8,500nucleotides, about 3,000 nucleotides to about 8,000 nucleotides, about3,000 nucleotides to about 7,800 nucleotides, about 3,000 nucleotides toabout 7,600 nucleotides, about 3,000 nucleotides to about 7,400nucleotides, about 3,000 nucleotides to about 7,200 nucleotides, about3,000 nucleotides to about 7,000 nucleotides, about 3,000 nucleotides toabout 6,800 nucleotides, about 3,000 nucleotides to about 6,600nucleotides, about 3,000 nucleotides to about 6,400 nucleotides, about3,000 nucleotides to about 6,200 nucleotides, about 3,000 nucleotides toabout 6,000 nucleotides, about 3,000 nucleotides to about 5,800nucleotides, about 3,000 nucleotides to about 5,600 nucleotides, about3,000 nucleotides to about 5,400 nucleotides, about 3,000 nucleotides toabout 5,200 nucleotides, about 3,000 nucleotides to about 5,000nucleotides, about 3,000 nucleotides to about 4,800 nucleotides, about3,000 nucleotides to about 4,600 nucleotides, about 3,000 nucleotides toabout 4,400 nucleotides, about 3,000 nucleotides to about 4,200nucleotides, about 3,000 nucleotides to about 4,000 nucleotides, about3,000 nucleotides to about 3,800 nucleotides, about 3,000 nucleotides toabout 3,600 nucleotides, about 3,000 nucleotides to about 3,400nucleotides, about 3,000 nucleotides to about 3,200 nucleotides, about3,200 nucleotides to about 10,000 nucleotides, about 3,200 nucleotidesto about 9,500 nucleotides, about 3,200 nucleotides to about 9,000nucleotides, about 3,200 nucleotides to about 8,500 nucleotides, about3,200 nucleotides to about 8,000 nucleotides, about 3,200 nucleotides toabout 7,800 nucleotides, about 3,200 nucleotides to about 7,600nucleotides, about 3,200 nucleotides to about 7,400 nucleotides, about3,200 nucleotides to about 7,200 nucleotides, about 3,200 nucleotides toabout 7,000 nucleotides, about 3,200 nucleotides to about 6,800nucleotides, about 3,200 nucleotides to about 6,600 nucleotides, about3,200 nucleotides to about 6,400 nucleotides, about 3,200 nucleotides toabout 6,200 nucleotides, about 3,200 nucleotides to about 6,000nucleotides, about 3,200 nucleotides to about 5,800 nucleotides, about3,200 nucleotides to about 5,600 nucleotides, about 3,200 nucleotides toabout 5,400 nucleotides, about 3,200 nucleotides to about 5,200nucleotides, about 3,200 nucleotides to about 5,000 nucleotides, about3,200 nucleotides to about 4,800 nucleotides, about 3,200 nucleotides toabout 4,600 nucleotides, about 3,200 nucleotides to about 4,400nucleotides, about 3,200 nucleotides to about 4,200 nucleotides, about3,200 nucleotides to about 4,000 nucleotides, about 3,200 nucleotides toabout 3,800 nucleotides, about 3,200 nucleotides to about 3,600nucleotides, about 3,200 nucleotides to about 3,400 nucleotides, about3,400 nucleotides to about 10,000 nucleotides, about 3,400 nucleotidesto about 9,500 nucleotides, about 3,400 nucleotides to about 9,000nucleotides, about 3,400 nucleotides to about 8,500 nucleotides, about3,400 nucleotides to about 8,000 nucleotides, about 3,400 nucleotides toabout 7,800 nucleotides, about 3,400 nucleotides to about 7,600nucleotides, about 3,400 nucleotides to about 7,400 nucleotides, about3,400 nucleotides to about 7,200 nucleotides, about 3,400 nucleotides toabout 7,000 nucleotides, about 3,400 nucleotides to about 6,800nucleotides, about 3,400 nucleotides to about 6,600 nucleotides, about3,400 nucleotides to about 6,400 nucleotides, about 3,400 nucleotides toabout 6,200 nucleotides, about 3,400 nucleotides to about 6,000nucleotides, about 3,400 nucleotides to about 5,800 nucleotides, about3,400 nucleotides to about 5,600 nucleotides, about 3,400 nucleotides toabout 5,400 nucleotides, about 3,400 nucleotides to about 5,200nucleotides, about 3,400 nucleotides to about 5,000 nucleotides, about3,400 nucleotides to about 4,800 nucleotides, about 3,400 nucleotides toabout 4,600 nucleotides, about 3,400 nucleotides to about 4,400nucleotides, about 3,400 nucleotides to about 4,200 nucleotides, about3,400 nucleotides to about 4,000 nucleotides, about 3,400 nucleotides toabout 3,800 nucleotides, about 3,400 nucleotides to about 3,600nucleotides, about 3,600 nucleotides to about 10,000 nucleotides, about3,600 nucleotides to about 9,500 nucleotides, about 3,600 nucleotides toabout 9,000 nucleotides, about 3,600 nucleotides to about 8,500nucleotides, about 3,600 nucleotides to about 8,000 nucleotides, about3,600 nucleotides to about 7,800 nucleotides, about 3,600 nucleotides toabout 7,600 nucleotides, about 3,600 nucleotides to about 7,400nucleotides, about 3,600 nucleotides to about 7,200 nucleotides, about3,600 nucleotides to about 7,000 nucleotides, about 3,600 nucleotides toabout 6,800 nucleotides, about 3,600 nucleotides to about 6,600nucleotides, about 3,600 nucleotides to about 6,400 nucleotides, about3,600 nucleotides to about 6,200 nucleotides, about 3,600 nucleotides toabout 6,000 nucleotides, about 3,600 nucleotides to about 5,800nucleotides, about 3,600 nucleotides to about 5,600 nucleotides, about3,600 nucleotides to about 5,400 nucleotides, about 3,600 nucleotides toabout 5,200 nucleotides, about 3,600 nucleotides to about 5,000nucleotides, about 3,600 nucleotides to about 4,800 nucleotides, about3,600 nucleotides to about 4,600 nucleotides, about 3,600 nucleotides toabout 4,400 nucleotides, about 3,600 nucleotides to about 4,200nucleotides, about 3,600 nucleotides to about 4,000 nucleotides, about3,600 nucleotides to about 3,800 nucleotides, about 3,800 nucleotides toabout 10,000 nucleotides, about 3,800 nucleotides to about 9,500nucleotides, about 3,800 nucleotides to about 9,000 nucleotides, about3,800 nucleotides to about 8,500 nucleotides, about 3,800 nucleotides toabout 8,000 nucleotides, about 3,800 nucleotides to about 7,800nucleotides, about 3,800 nucleotides to about 7,600 nucleotides, about3,800 nucleotides to about 7,400 nucleotides, about 3,800 nucleotides toabout 7,200 nucleotides, about 3,800 nucleotides to about 7,000nucleotides, about 3,800 nucleotides to about 6,800 nucleotides, about3,800 nucleotides to about 6,600 nucleotides, about 3,800 nucleotides toabout 6,400 nucleotides, about 3,800 nucleotides to about 6,200nucleotides, about 3,800 nucleotides to about 6,000 nucleotides, about3,800 nucleotides to about 5,800 nucleotides, about 3,800 nucleotides toabout 5,600 nucleotides, about 3,800 nucleotides to about 5,400nucleotides, about 3,800 nucleotides to about 5,200 nucleotides, about3,800 nucleotides to about 5,000 nucleotides, about 3,800 nucleotides toabout 4,800 nucleotides, about 3,800 nucleotides to about 4,600nucleotides, about 3,800 nucleotides to about 4,200 nucleotides, about3,800 nucleotides to about 4,000 nucleotides, about 4,000 nucleotides toabout 10,000 nucleotides, about 4,000 nucleotides to about 9,500nucleotides, about 4,000 nucleotides to about 9,000 nucleotides, about4,000 nucleotides to about 8,500 nucleotides, about 4,000 nucleotides toabout 8,000 nucleotides, about 4,000 nucleotides to about 7,800nucleotides, about 4,000 nucleotides to about 7,600 nucleotides, about4,000 nucleotides to about 7,400 nucleotides, about 4,000 nucleotides toabout 7,200 nucleotides, about 4,000 nucleotides to about 7,000nucleotides, about 4,000 nucleotides to about 6,800 nucleotides, about4,000 nucleotides to about 6,600 nucleotides, about 4,000 nucleotides toabout 6,400 nucleotides, about 4,000 nucleotides to about 6,200nucleotides, about 4,000 nucleotides to about 6,000 nucleotides, about4,000 nucleotides to about 5,800 nucleotides, about 4,000 nucleotides toabout 5,600 nucleotides, about 4,000 nucleotides to about 5,400nucleotides, about 4,000 nucleotides to about 5,200 nucleotides, about4,000 nucleotides to about 5,000 nucleotides, about 4,000 nucleotides toabout 4,800 nucleotides, about 4,000 nucleotides to about 4,600nucleotides, about 4,000 nucleotides to about 4,400 nucleotides, about4,000 nucleotides to about 4,200 nucleotides, about 4,200 nucleotides toabout 10,000 nucleotides, about 4,200 nucleotides to about 9,500nucleotides, about 4,200 nucleotides to about 9,000 nucleotides, about4,200 nucleotides to about 8,500 nucleotides, about 4,200 nucleotides toabout 8,000 nucleotides, about 4,200 nucleotides to about 7,800nucleotides, about 4,200 nucleotides to about 7,600 nucleotides, about4,200 nucleotides to about 7,400 nucleotides, about 4,200 nucleotides toabout 7,200 nucleotides, about 4,200 nucleotides to about 7,000nucleotides, about 4,200 nucleotides to about 6,800 nucleotides, about4,200 nucleotides to about 6,600 nucleotides, about 4,200 nucleotides toabout 6,400 nucleotides, about 4,200 nucleotides to about 6,200nucleotides, about 4,200 nucleotides to about 6,000 nucleotides, about4,200 nucleotides to about 5,800 nucleotides, about 4,200 nucleotides toabout 5,600 nucleotides, about 4,200 nucleotides to about 5,400nucleotides, about 4,200 nucleotides to about 5,200 nucleotides, about4,200 nucleotides to about 5,000 nucleotides, about 4,200 nucleotides toabout 4,800 nucleotides, about 4,200 nucleotides to about 4,600nucleotides, about 4,200 nucleotides to about 4,400 nucleotides, about4,400 nucleotides to about 10,000 nucleotides, about 4,400 nucleotidesto about 9,500 nucleotides, about 4,400 nucleotides to about 9,000nucleotides, about 4,400 nucleotides to about 8,500 nucleotides, about4,400 nucleotides to about 8,000 nucleotides, about 4,400 nucleotides toabout 7,800 nucleotides, about 4,400 nucleotides to about 7,600nucleotides, about 4,400 nucleotides to about 7,400 nucleotides, about4,400 nucleotides to about 7,200 nucleotides, about 4,400 nucleotides toabout 7,000 nucleotides, about 4,400 nucleotides to about 6,800nucleotides, about 4,400 nucleotides to about 6,600 nucleotides, about4,400 nucleotides to about 6,400 nucleotides, about 4,400 nucleotides toabout 6,200 nucleotides, about 4,400 nucleotides to about 6,000nucleotides, about 4,400 nucleotides to about 5,800 nucleotides, about4,400 nucleotides to about 5,600 nucleotides, about 4,400 nucleotides toabout 5,400 nucleotides, about 4,400 nucleotides to about 5,200nucleotides, about 4,400 nucleotides to about 5,000 nucleotides, about4,400 nucleotides to about 4,800 nucleotides, about 4,400 nucleotides toabout 4,600 nucleotides, about 4,600 nucleotides to about 10,000nucleotides, about 4,600 nucleotides to about 9,500 nucleotides, about4,600 nucleotides to about 9,000 nucleotides, about 4,600 nucleotides toabout 8,500 nucleotides, about 4,600 nucleotides to about 8,000nucleotides, about 4,600 nucleotides to about 7,800 nucleotides, about4,600 nucleotides to about 7,600 nucleotides, about 4,600 nucleotides toabout 7,400 nucleotides, about 4,600 nucleotides to about 7,200nucleotides, about 4,600 nucleotides to about 7,000 nucleotides, about4,600 nucleotides to about 6,800 nucleotides, about 4,600 nucleotides toabout 6,600 nucleotides, about 4,600 nucleotides to about 6,400nucleotides, about 4,600 nucleotides to about 6,200 nucleotides, about4,600 nucleotides to about 6,000 nucleotides, about 4,600 nucleotides toabout 5,800 nucleotides, about 4,600 nucleotides to about 5,600nucleotides, about 4,600 nucleotides to about 5,400 nucleotides, about4,600 nucleotides to about 5,200 nucleotides, about 4,600 nucleotides toabout 5,000 nucleotides, about 4,600 nucleotides to about 4,800nucleotides, about 4,800 nucleotides to about 10,000 nucleotides, about4,800 nucleotides to about 9,500 nucleotides, about 4,800 nucleotides toabout 9,000 nucleotides, about 4,800 nucleotides to about 8,500nucleotides, about 4,800 nucleotides to about 8,000 nucleotides, about4,800 nucleotides to about 7,800 nucleotides, about 4,800 nucleotides toabout 7,600 nucleotides, about 4,800 nucleotides to about 7,400nucleotides, about 4,800 nucleotides to about 7,200 nucleotides, about4,800 nucleotides to about 7,000 nucleotides, about 4,800 nucleotides toabout 6,800 nucleotides, about 4,800 nucleotides to about 6,600nucleotides, about 4,800 nucleotides to about 6,400 nucleotides, about4,800 nucleotides to about 6,200 nucleotides, about 4,800 nucleotides toabout 6,000 nucleotides, about 4,800 nucleotides to about 5,800nucleotides, about 4,800 nucleotides to about 5,600 nucleotides, about4,800 nucleotides to about 5,400 nucleotides, about 4,800 nucleotides toabout 5,200 nucleotides, about 4,800 nucleotides to about 5,000nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about5,000 nucleotides to about 9,500 nucleotides, about 5,000 nucleotides toabout 9,000 nucleotides, about 5,000 nucleotides to about 8,500nucleotides, about 5,000 nucleotides to about 8,000 nucleotides, about5,000 nucleotides to about 7,800 nucleotides, about 5,000 nucleotides toabout 7,600 nucleotides, about 5,000 nucleotides to about 7,400nucleotides, about 5,000 nucleotides to about 7,200 nucleotides, about5,000 nucleotides to about 7,000 nucleotides, about 5,000 nucleotides toabout 6,800 nucleotides, about 5,000 nucleotides to about 6,600nucleotides, about 5,000 nucleotides to about 6,400 nucleotides, about5,000 nucleotides to about 6,200 nucleotides, about 5,000 nucleotides toabout 6,000 nucleotides, about 5,000 nucleotides to about 5,800nucleotides, about 5,000 nucleotides to about 5,600 nucleotides, about5,000 nucleotides to about 5,400 nucleotides, about 5,000 nucleotides toabout 5,200 nucleotides, about 5,200 nucleotides to about 10,000nucleotides, about 5,200 nucleotides to about 9,500 nucleotides, about5,200 nucleotides to about 9,000 nucleotides, about 5,200 nucleotides toabout 8,500 nucleotides, about 5,200 nucleotides to about 8,000nucleotides, about 5,200 nucleotides to about 7,800 nucleotides, about5,200 nucleotides to about 7,600 nucleotides, about 5,200 nucleotides toabout 7,400 nucleotides, about 5,200 nucleotides to about 7,200nucleotides, about 5,200 nucleotides to about 7,000 nucleotides, about5,200 nucleotides to about 6,800 nucleotides, about 5,200 nucleotides toabout 6,600 nucleotides, about 5,200 nucleotides to about 6,400nucleotides, about 5,200 nucleotides to about 6,200 nucleotides, about5,200 nucleotides to about 6,000 nucleotides, about 5,200 nucleotides toabout 5,800 nucleotides, about 5,200 nucleotides to about 5,600nucleotides, about 5,200 nucleotides to about 5,400 nucleotides, about5,400 nucleotides to about 10,000 nucleotides, about 5,400 nucleotidesto about 9,500 nucleotides, about 5,400 nucleotides to about 9,000nucleotides, about 5,400 nucleotides to about 8,500 nucleotides, about5,400 nucleotides to about 8,000 nucleotides, about 5,400 nucleotides toabout 7,800 nucleotides, about 5,400 nucleotides to about 7,600nucleotides, about 5,400 nucleotides to about 7,400 nucleotides, about5,400 nucleotides to about 7,200 nucleotides, about 5,400 nucleotides toabout 7,000 nucleotides, about 5,400 nucleotides to about 6,800nucleotides, about 5,400 nucleotides to about 6,600 nucleotides, about5,400 nucleotides to about 6,400 nucleotides, about 5,400 nucleotides toabout 6,200 nucleotides, about 5,400 nucleotides to about 6,000nucleotides, about 5,400 nucleotides to about 5,800 nucleotides, about5,400 nucleotides to about 5,600 nucleotides, about 5,600 nucleotides toabout 10,000 nucleotides, about 5,600 nucleotides to about 9,500nucleotides, about 5,600 nucleotides to about 9,000 nucleotides, about5,600 nucleotides to about 8,500 nucleotides, about 5,600 nucleotides toabout 8,000 nucleotides, about 5,600 nucleotides to about 7,800nucleotides, about 5,600 nucleotides to about 7,600 nucleotides, about5,600 nucleotides to about 7,400 nucleotides, about 5,600 nucleotides toabout 7,200 nucleotides, about 5,600 nucleotides to about 7,000nucleotides, about 5,600 nucleotides to about 6,800 nucleotides, about5,600 nucleotides to about 6,600 nucleotides, about 5,600 nucleotides toabout 6,400 nucleotides, about 5,600 nucleotides to about 6,200nucleotides, about 5,600 nucleotides to about 6,000 nucleotides, about5,600 nucleotides to about 5,800 nucleotides, about 5,800 nucleotides toabout 10,000 nucleotides, about 5,800 nucleotides to about 9,500nucleotides, about 5,800 nucleotides to about 9,000 nucleotides, about5,800 nucleotides to about 8,500 nucleotides, about 5,800 nucleotides toabout 8,000 nucleotides, about 5,800 nucleotides to about 7,800nucleotides, about 5,800 nucleotides to about 7,600 nucleotides, about5,800 nucleotides to about 7,400 nucleotides, about 5,800 nucleotides toabout 7,200 nucleotides, about 5,800 nucleotides to about 7,000nucleotides, about 5,800 nucleotides to about 6,800 nucleotides, about5,800 nucleotides to about 6,600 nucleotides, about 5,800 nucleotides toabout 6,400 nucleotides, about 5,800 nucleotides to about 6,200nucleotides, about 5,800 nucleotides to about 6,000 nucleotides, about6,000 nucleotides to about 10,000 nucleotides, about 6,000 nucleotidesto about 9,500 nucleotides, about 6,000 nucleotides to about 9,000nucleotides, about 6,000 nucleotides to about 8,500 nucleotides, about6,000 nucleotides to about 8,000 nucleotides, about 6,000 nucleotides toabout 7,800 nucleotides, about 6,000 nucleotides to about 7,600nucleotides, about 6,000 nucleotides to about 7,400 nucleotides, about6,000 nucleotides to about 7,200 nucleotides, about 6,000 nucleotides toabout 7,000 nucleotides, about 6,000 nucleotides to about 6,800nucleotides, about 6,000 nucleotides to about 6,600 nucleotides, about6,000 nucleotides to about 6,400 nucleotides, about 6,000 nucleotides toabout 6,200 nucleotides, about 6,200 nucleotides to about 10,000nucleotides, about 6,200 nucleotides to about 9,000 nucleotides, about6,200 nucleotides to about 8,500 nucleotides, about 6,200 nucleotides toabout 8,000 nucleotides, about 6,200 nucleotides to about 7,800nucleotides, about 6,200 nucleotides to about 7,600 nucleotides, about6,200 nucleotides to about 7,400 nucleotides, about 6,200 nucleotides toabout 7,200 nucleotides, about 6,200 nucleotides to about 7,000nucleotides, about 6,200 nucleotides to about 6,800 nucleotides, about6,200 nucleotides to about 6,600 nucleotides, about 6,200 nucleotides toabout 6,400 nucleotides, about 6,400 nucleotides to about 10,000nucleotides, about 6,400 nucleotides to about 9,500 nucleotides, about6,400 nucleotides to about 9,000 nucleotides, about 6,400 nucleotides toabout 8,500 nucleotides, about 6,400 nucleotides to about 8,000nucleotides, about 6,400 nucleotides to about 7,800 nucleotides, about6,400 nucleotides to about 7,600 nucleotides, about 6,400 nucleotides toabout 7,400 nucleotides, about 6,400 nucleotides to about 7,200nucleotides, about 6,400 nucleotides to about 7,000 nucleotides, about6,400 nucleotides to about 6,800 nucleotides, about 6,400 nucleotides toabout 6,600 nucleotides, about 6,600 nucleotides to about 10,000nucleotides, about 6,600 nucleotides to about 9,500 nucleotides, about6,600 nucleotides to about 9,000 nucleotides, about 6,600 nucleotides toabout 8,500 nucleotides, about 6,600 nucleotides to about 8,000nucleotides, about 6,600 nucleotides to about 7,800 nucleotides, about6,600 nucleotides to about 7,600 nucleotides, about 6,600 nucleotides toabout 7,400 nucleotides, about 6,600 nucleotides to about 7,200nucleotides, about 6,600 nucleotides to about 7,000 nucleotides, about6,600 nucleotides to about 6,800 nucleotides, about 6,800 nucleotides toabout 10,000 nucleotides, about 6,800 nucleotides to about 9,500nucleotides, about 6,800 nucleotides to about 9,000 nucleotides, about6,800 nucleotides to about 8,500 nucleotides, about 6,800 nucleotides toabout 8,000 nucleotides, about 6,800 nucleotides to about 7,800nucleotides, about 6,800 nucleotides to about 7,600 nucleotides, about6,800 nucleotides to about 7,400 nucleotides, about 6,800 nucleotides toabout 7,200 nucleotides, about 6,800 nucleotides to about 7,000nucleotides, about 7,000 nucleotides to about 10,000 nucleotides, about7,000 nucleotides to about 9,500 nucleotides, about 7,000 nucleotides toabout 9,000 nucleotides, about 7,000 nucleotides to about 8,500nucleotides, about 7,000 nucleotides to about 8,000 nucleotides, about7,000 nucleotides to about 7,800 nucleotides, about 7,000 nucleotides toabout 7,600 nucleotides, about 7,000 nucleotides to about 7,400nucleotides, about 7,000 nucleotides to about 7,200 nucleotides, about7,200 nucleotides to about 10,000 nucleotides, about 7,200 nucleotidesto about 9,500 nucleotides, about 7,200 nucleotides to about 9,000nucleotides, about 7,200 nucleotides to about 8,500 nucleotides, about7,200 nucleotides to about 8,000 nucleotides, about 7,200 nucleotides toabout 7,800 nucleotides, about 7,200 nucleotides to about 7,600nucleotides, about 7,200 nucleotides to about 7,400 nucleotides, about7,400 nucleotides to about 10,000 nucleotides, about 7,400 nucleotidesto about 9,500 nucleotides, about 7,400 nucleotides to about 9,000nucleotides, about 7,400 nucleotides to about 8,500 nucleotides, about7,400 nucleotides to about 8,000 nucleotides, about 7,400 nucleotides toabout 7,800 nucleotides, about 7,400 nucleotides to about 7,600nucleotides, about 7,600 nucleotides to about 10,000 nucleotides, about7,600 nucleotides to about 9,500 nucleotides, about 7,600 nucleotides toabout 9,000 nucleotides, about 7,600 nucleotides to about 8,500nucleotides, about 7,600 nucleotides to about 8,000 nucleotides, about7,600 nucleotides to about 7,800 nucleotides, about 7,800 nucleotides toabout 10,000 nucleotides, about 7,800 nucleotides to about 9,500nucleotides, about 7,800 nucleotides to about 9,000 nucleotides, about7,800 nucleotides to about 8,500 nucleotides, about 7,800 nucleotides toabout 8,000 nucleotides, about 8,000 nucleotides to about 10,000nucleotides, about 8,000 nucleotides to about 9,500 nucleotides, about8,000 nucleotides to about 9,000 nucleotides, about 8,000 nucleotides toabout 8,500 nucleotides, about 8,500 nucleotides to about 10,000nucleotides, about 8,500 nucleotides to about 9,500 nucleotides, about8,500 nucleotides to about 9,000 nucleotides, about 9,000 nucleotides toabout 10,000 nucleotides, about 9,000 nucleotides to about 9,500nucleotides, or about 9,500 nucleotides to about 10,000 nucleotides(inclusive).

A variety of different methods known in the art can be used to introduceany of the vectors disclosed herein into a mammalian cell (e.g., acochlear inner hair cell). Non-limiting examples of methods forintroducing nucleic acid into a mammalian cell include: lipofection,transfection (e.g., calcium phosphate transfection, transfection usinghighly branched organic compounds, transfection using cationic polymers,dendrimer-based transfection, optical transfection, particle-basedtransfection (e.g., nanoparticle transfection), or transfection usingliposomes (e.g., cationic liposomes)), microinjection, electroporation,cell squeezing, sonoporation, protoplast fusion, impalefection,hydrodynamic delivery, gene gun, magnetofection, viral transfection, andnucleofection.

Skilled practitioners will appreciate that any of the vectors describedherein can be introduced into a mammalian cell by, for example,lipofection.

Various molecular biology techniques that can be used to introduce amutation(s) and/or a deletion(s) into an endogenous gene are also knownin the art. Non-limiting examples of such techniques includesite-directed mutagenesis, CRISPR (e.g., CRISPR/Cas9-induced knock-inmutations and CRISPR/Cas9-induced knock-out mutations), and TALENs.These methods can be used to correct the sequence of a defectiveendogenous gene present in a chromosome of a target cell.

Any of the vectors described herein can further include a controlsequence, e.g., a control sequence selected from the group of atranscription initiation sequence, a transcription termination sequence,a promoter sequence, an enhancer sequence, an RNA splicing sequence, apolyadenylation (polyA) sequence, and a Kozak consensus sequence.Non-limiting examples of these control sequences are described herein.In some embodiments, a promoter can be a native promoter, a constitutivepromoter, an inducible promoter, and/or a tissue-specific promoter.

Promoters

Non-limiting examples of promoters are described herein. Additionalexamples of promoters are known in the art.

In some embodiments, a vector encoding an N-terminal portion of anotoferlin protein (e.g., a human otoferlin protein) can include apromoter and/or an enhancer. The vector encoding the N-terminal portionof the otoferlin protein can include any of the promoters and/orenhancers described herein or known in the art.

In some embodiments, the promoter is an inducible promoter, aconstitutive promoter, a mammalian cell promoter, a viral promoter, achimeric promoter, an engineered promoter, a tissue-specific promoter,or any other type of promoter known in the art. In some embodiments, thepromoter is a RNA polymerase II promoter, such as a mammalian RNApolymerase II promoter. In some embodiments, the promoter is a RNApolymerase III promoter, including, but not limited to, a H1 promoter, ahuman U6 promoter, a mouse U6 promoter, or a swine U6 promoter. Thepromoter will generally be one that is able to promote transcription incochlear cells such as hair cells. In some examples, the promoter is acochlea-specific promoter or a cochlea-oriented promoter.

A variety of promoters are known in the art that can be used herein.Non-limiting examples of promoters that can be used herein include:human elongation factor 1α-subunit (EF1a) (Liu et al. (2007) Exp. Mol.Med. 39(2): 170-175; Accession No. J04617.1; Gill et al., Gene Ther.8(20):1539-1546, 2001; Xu et al., Human Gene Ther. 12(5):563-573, 2001;Xu et al., Gene Ther. 8:1323-1332; Ikeda et al., Gene Ther. 9:932-938,2002; Gilham et al., J. Gene Med. 12(2):129-136, 2010), cytomegalovirus(Xu et al., Human Gene Ther. 12(5):563-573, 2001; Xu et al., Gene Ther.8:1323-1332; Gray et al., Human Gene Ther. 22:1143-1153, 2011), humanimmediate-early cytomegalovirus (CMV) (U.S. Pat. No. 5,168,062, Liu etal. (2007) Exp. Mol. Med. 39(2): 170-175; Accession No. X17403.1 orKY490085.1), human ubiquitin C (UBC) (Gill et al., Gene Ther.8(20):1539-1546, 2001; Qin et al., PLoS One 5(5):e10611, 2010), mousephosphoglycerate kinase 1, polyoma adenovirus, simian virus 40 (SV40),β-globin, p-actin, α-fetoprotein, γ-globin, β-interferon, γ-glutamyltransferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, ratinsulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II(MT II), amylase, cathepsin, MI muscarinic receptor, retroviral LTR(e.g. human T-cell leukemia virus HTLV), AAV ITR, interleukin-2,collagenase, platelet-derived growth factor, adenovirus 5 E2,stromelysin, murine MX gene, glucose regulated proteins (GRP78 andGRP94), α-2-macroglobulin, vimentin, MHC class I gene H-2κ b, HSP70,proliferin, tumor necrosis factor, thyroid stimulating hormone α gene,immunoglobulin light chain, T-cell receptor, HLA DQa and DQP,interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, musclecreatine kinase, prealbumin (transthyretin), elastase I, albumin gene,c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B)histone, rat growth hormone, human serum amyloid (SAA), troponin I (TNI), duchenne muscular dystrophy, human immunodeficiency virus, GibbonApe Leukemia Virus (GALV) promoters, promoter of HNRPA2B1-CBX1 (UCOE)(Powell and Gray (2015) Discov. Med. 19(102): 49-57; Antoniou et al.,Human Gene Ther. 24(4):363-374, 2013), β-glucuronidase (GUSB) (Husain etal., Gene Ther. 16:927-932, 2009), chicken β-actin (CBA) (Liu et al.(2007) Exp. Mol. Med. 39(2): 170-175; Stone et al. (2005) Mol. Ther.11(6): 843-848; Klein et al., Exp. Neurol. 176(1):66-74, 2002; Ohlfestet al., Blood 105:2691-2698, 2005; Gray et al., Human Gene Ther.22:1143-1153, 2011), a human β-actin promoter (HBA) (Accession No.Y00474.1), murine myosin VIIA (musMyo7) (Boeda et al. (2001) Hum. Mol.Genet. 10(15): 1581-1589; Accession No. AF384559.1), human myosin VIIA(hsMyo7) (Boeda et al. (2001) Hum. Mol. Genet. 10(15): 1581-1589;Accession No. NG_009086.1), murine poly(ADP-ribose) polymerase 2(musPARP2) (Ame et al. (2001) J. Biol. Chem. 276(14): 11092-11099;Accession No. AF191547.1), human poly(ADP-ribose) polymerase 2 (hsPARP2)(Ame et al. (2001) J. Biol. Chem. 276(14): 11092-11099; Accession No.X16612.1 or AF479321.1), acetylcholine receptor epsilon-subunit (AChε)(Duclert et al. (1993) PNAS 90(7): 3043-3047; Accession No. S58221.1 orCR933736.12), Rous sarcoma virus (RSV) (Liu et al. (2007) Exp. Mol. Med.39(2): 170-175; Accession No. M77786.1), (GFAP) (Liu et al. (2007) Exp.Mol. Med. 39(2): 170-175; Stone et al. (2005) Mol. Ther. 11(6): 843-848;Accession No. NG_008401.1 or M67446.1), hAAT (Van Linthout et al., HumanGene Ther. 13(7):829-840, 2002; Cunningham et al., Mol. Ther.16(6):1081-1088, 2008), and a CBA hybrid (CBh) (Gray et al. (2011) Hum.Gen. Therapy 22: 1143-1153; Accession No. KF926476.1 or KC152483.1).Additional examples of promoters are known in the art. See, e.g.,Lodish, Molecular Cell Biology, Freeman and Company, New York 2007. Insome embodiments, the promoter is the CMV immediate early promoter.

In some embodiments, the promoter is a CAG promoter or a CAG/CBApromoter. In some embodiments, a vector or construct of the presentdisclosure comprises a CAG promoter. In some embodiments, a CAG promotercomprises, in order from 5′ to 3′, the nucleotide sequences of SEQ IDNOs: 98, 99, and 100. In some such embodiments, a CAG promoter comprisesa CMV early enhancer element (e.g., SEQ ID NO: 98), a chicken beta actin(CBA) gene sequence (e.g., SEQ ID NO: 99), and a chimeric intron/3′splice sequence from the rabbit beta globin gene (e.g., SEQ ID NO: 100).

The term “operably linked” or “transcriptional control” refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Operably linked DNA sequences can be contiguous with each other and,e.g., where necessary to join two protein coding regions, are in thesame reading frame.

The term “constitutive” promoter refers to a nucleotide sequence that,when operably linked with a nucleic acid encoding a protein (e.g., anotoferlin protein), causes RNA to be transcribed from the nucleic acidin a mammalian cell under most or all physiological conditions.

The term “constitutive” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cell undermost or all physiological conditions of the cell.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus(CMV) promoter (see, e.g., Boshart et al. Cell 41:521-530, 1985), theSV40 promoter, the dihydrofolate reductase promoter, the beta-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1-alphapromoter (Invitrogen).

The term “inducible” promoter refers to a nucleotide sequence which,when operably linked with a polynucleotide which encodes or specifies agene product, causes the gene product to be produced in a cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech, and Ariad. Additional examples ofinducible promoters are known in the art.

Examples of inducible promoters regulated by exogenously suppliedcompounds include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); theecdysone insect promoter (No et al. Proc. Natl. Acad. Sci. U.S.A.93:3346-3351, 1996), the tetracycline-repressible system (Gossen et al.Proc. Natl. Acad. Sci. U.S.A. 89:5547-5551, 1992), thetetracycline-inducible system (Gossen et al. Science 268:1766-1769,1995, see also Harvey et al. Curr. Opin. Chem. Biol. 2:512-518, 1998),the RU486-inducible system (Wang et al. Nat. Biotech. 15:239-243, 1997)and Wang et al. Gene Ther. 4:432-441, 1997), and the rapamycin-induciblesystem (Magari et al. J. Clin. Invest. 100:2865-2872, 1997).

The term “tissue-specific” promoter refers to a promoter that is activeonly in certain specific cell types and/or tissues (e.g., transcriptionof a specific gene occurs only within cells expressing transcriptionregulatory proteins that bind to the tissue-specific promoter).

The term “tissue-specific” promoter refers to a nucleotide sequencewhich, when operably linked with a polynucleotide encodes or specifiedby a gene, causes the gene product to be produced in a cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

The term “regulatory sequence” refers to a nucleic acid sequence whichis regulates expression of a gene product operably linked to theregulatory sequence. In some instances, this sequence may be an enhancersequence and other regulatory elements which regulate expression of thegene product.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue-specific manner.

In some embodiments, the tissue-specific promoter is a cochlea-specificpromoter. In some embodiments, the tissue-specific promoter is acochlear hair cell-specific promoter. Non-limiting examples of cochlearhair cell-specific promoters include but are not limited to: a ATOH1promoter, a POU4F3 promoter, a LHX3 promoter, a MYO7A promoter, a MYO6promoter, a α9ACHR promoter, and a α10ACHR promoter.

Enhancers and 5′ Cap

In some instances, a vector can include a promoter sequence and/or anenhancer sequence. The term “enhancer” refers to a nucleotide sequencethat can increase the level of transcription of a nucleic acid encodinga protein of interest (e.g., an otoferlin protein), Enhancer sequences(50-1500 base pairs in length) generally increase the level oftranscription by providing additional binding sites fortranscription-associated proteins (e.g., transcription factors). In someembodiments, an enhancer sequence is found within an intronic sequence.Unlike promoter sequences, enhancer sequences can act at much largerdistance away from the transcription start site (e.g., as compared to apromoter). Non-limiting examples of enhancers include a RSV enhancer, aCMV enhancer, and a SV40 enhancer. An example of a CMV enhancer isdescribed in, e.g., Boshart et al., Cell 41(2):521-530, 1985.

As used herein, a 5′ cap (also termed an RNA cap, an RNA7-methylguanosine cap or an RNA m.sup.7G cap) is a modified guaninenucleotide that has been added to the “front” or 5′ end of a eukaryoticmessenger RNA shortly after the start of transcription. The 5′ capconsists of a terminal group which is linked to the first transcribednucleotide. Its presence is critical for recognition by the ribosome andprotection from RNases. Cap addition is coupled to transcription, andoccurs co-transcriptionally, such that each influences the other.Shortly after the start of transcription, the 5′ end of the mRNA beingsynthesized is bound by a cap-synthesizing complex associated with RNApolymerase. This enzymatic complex catalyzes the chemical reactions thatare required for mRNA capping. Synthesis proceeds as a multi-stepbiochemical reaction. The capping moiety can be modified to modulatefunctionality of mRNA such as its stability or efficiency oftranslation.

Poly(A) Sequences

In some embodiments, any of the vectors provided herein can include apoly(A) sequence. Most nascent eukaryotic mRNAs possess a poly(A) tailat their 3′ end which is added during a complex process that includescleavage of the primary transcript and a coupled polyadenylationreaction (see, e.g., Proudfoot et al., Cell 108:501-512, 2002). Thepoly(A) tail confers mRNA stability and transferability (MolecularBiology of the Cell, Third Edition by B. Alberts et al., GarlandPublishing, 1994). In some embodiments, the poly(A) sequence ispositioned 3′ to the nucleic acid sequence encoding the C-terminus ofthe otoferlin protein.

As used herein, “polyadenylation” refers to the covalent linkage of apolyadenylyl moiety, or its modified variant, to a messenger RNAmolecule. In eukaryotic organisms, most messenger RNA (mRNA) moleculesare polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequenceof adenine nucleotides (often several hundred) added to the pre-mRNAthrough the action of an enzyme, polyadenylate polymerase. In highereukaryotes, the poly(A) tail is added onto transcripts that contain aspecific sequence, the polyadenylation signal. The poly(A) tail and theprotein bound to it aid in protecting mRNA from degradation byexonucleases. Polyadenylation is also important for transcriptiontermination, export of the mRNA from the nucleus, and translation.Polyadenylation occurs in the nucleus immediately after transcription ofDNA into RNA, but additionally can also occur later in the cytoplasm.After transcription has been terminated, the mRNA chain is cleavedthrough the action of an endonuclease complex associated with RNApolymerase. The cleavage site is usually characterized by the presenceof the base sequence AAUAAA near the cleavage site. After the mRNA hasbeen cleaved, adenosine residues are added to the free 3′ end at thecleavage site.

As used herein, a “poly(A) signal sequence” is a sequence that triggersthe endonuclease cleavage of an mRNA and the additional of a series ofadenosines to the 3′ end of the cleaved mRNA.

As used herein, a “poly(A)” is a series of adenosines attached bypolyadenylation to the mRNA. In the preferred embodiment of a constructfor transient expression, the polyA is between 50 and 5000, preferablygreater than 64, more preferably greater than 100, most preferablygreater than 300 or 400. Poly(A) sequences can be modified chemically orenzymatically to modulate mRNA functionality such as localization,stability or efficiency of translation.

There are several poly(A) signal sequences that can be used, includingthose derived from bovine growth hormone (bgh) (Woychik et al., Proc.Natl. Acad. Sci. U.S.A. 81(13):3944-3948, 1984; U.S. Pat. No. 5,122,458;Yew et al., Human Gene Ther. 8(5):575-584, 1997; Xu et al., Human GeneTher. 12(5):563-573, 2001; Xu et al., Gene Ther. 8:1323-1332, 2001; Wuet al., Mol. Ther. 16(2):280-289, 2008; Gray et al., Human Gene Ther.22:1143-1153, 2011; Choi et al., Mol. Brain 7:17, 2014), mouse-p-globin,mouse-α-globin (Orkin et al., EMBO J. 4(2):453-456, 1985; Thein et al.,Blood 71(2):313-319, 1988), human collagen, polyoma virus (Batt et al.,Mol. Cell Biol. 15(9):4783-4790, 1995), the Herpes simplex virusthymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylationsignal (US 2006/0040354), human growth hormone (hGH) (Szymanski et al.,Mol. Therapy 15(7):1340-1347, 2007; Ostegaard et al., Proc. Natl. Acad.Sci. U.S.A. 102(8):2952-2957, 2005), synthetic polyA (Levitt et al.,Genes Dev. 3(7):1019-1025, 1989; Yew et al., Human Gene Ther.8(5):575-584, 1997; Ostegaard et al., Proc. Natl. Acad. Sci. U.S.A.102(8):2952-2957, 2005; Choi et al., Mol. Brain 7:17, 2014), HIV-1upstream poly(A) enhancer (Schambach et al., Mol. Ther. 15(6):1167-1173,2007), adenovirus (L3) upstream poly(A) enhancer (Schambach et al., Mol.Ther. 15(6):1167-1173, 2007), hTHGB upstream poly(A) enhancer (Schambachet al., Mol. Ther. 15(6):1167-1173, 2007), hC2 upstream poly(A) enhancer(Schambach et al., Mol. Ther. 15(6):1167-1173, 2007), the groupconsisting of SV40 poly(A) signal sequence, such as the SV40 late andearly poly(A) signal sequence (Schek et al., Mol. Cell Biol.12(12):5386-5393, 1992; Choi et al., Mol. Brain 7:17, 2014; Schambach etal., Mol. Ther. 15(6):1167-1173, 2007). A non-limiting example of apoly(A) signal sequence is SEQ ID NO: 68, 76, or 77.

The poly(A) signal sequence can be the sequence AATAAA. The AATAAAsequence may be substituted with other hexanucleotide sequences withhomology to AATAAA which are capable of signaling polyadenylation,including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA,AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA,AATTAA, or AATAAG (see, e.g., WO 06/12414).

In some embodiments, the poly(A) signal sequence can be a syntheticpolyadenylation site (see, e.g., the pCl-neo expression vector ofPromega which is based on Levitt el al, Genes Dev. 3(7):1019-1025,1989). In some embodiments, the poly(A) signal sequence is thepolyadenylation signal of soluble neuropilin-1 (sNRP)(AAATAAAATACGAAATG) (see, e.g., WO 05/073384). In some embodiments, apoly(A) sequence is a bovine growth hormone poly(A) sequence. In somesuch embodiments, a bGH poly(A) sequence comprises or is the sequence ofSEQ ID NO: 108. In some embodiments, a vector or construct of thepresent disclosure comprises a boving growth hormone polyA sequencerepresented by SEQ ID NO: 108. Additional examples of poly(A) signalsequences are known in the art.

In some embodiments, any of the vectors provided herein can include awoodchuck hepatitis virus posttranscriptional regulatory element (WPRE),e.g., SEQ ID NO: 69.

Internal Ribosome Entry Site (IRES)

In some embodiments, a vector encoding the C-terminus of the otoferlinprotein can include a polynucleotide internal ribosome entry site(IRES). An IRES sequence is used to produce more than one polypeptidefrom a single gene transcript. An IRES forms a complex secondarystructure that allows translation initiation to occur from any positionwith an mRNA immediately downstream from where the IRES is located (see,e.g., Pelletier and Sonenberg, Mol. Cell. Biol. 8(3):1103-1112, 1988).

There are several IRES sequences known to those skilled in the art,including those from, e.g., foot and mouth disease virus (FMDV),encephalomyocarditis virus (EMCV), human rhinovirus (HRV), cricketparalysis virus, human immunodeficiency virus (HIV), hepatitis A virus(HAV), hepatitis C virus (HCV), and poliovirus (PV). See e.g., Alberts,Molecular Biology of the Cell, Garland Science, 2002; and Hellen et al.,Genes Dev. 15(13):1593-612, 2001.

In some embodiments, the IRES sequence that is incorporated into thevector that encodes the C-terminus of an otoferlin protein is the footand mouth disease virus (FMDV). The Foot and Mouth Disease Virus 2Asequence is a small peptide (approximately 18 amino acids in length)that has been shown to mediate the cleavage of polyproteins (Ryan, M Det al., EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127,1996; Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al,Plant Journal 4:453-459, 1999). The cleavage activity of the 2A sequencehas previously been demonstrated in artificial systems includingplasmids and gene therapy vectors (AAV and retroviruses) (Ryan et al.,EMBO 4:928-933, 1994; Mattion et al., J. Virology 70:8124-8127, 1996;Furler et al., Gene Therapy 8:864-873, 2001; and Halpin et al., PlantJournal 4:453-459, 1999; de Felipe et al., Gene Therapy 6:198-208, 1999;de Felipe et al., Human Gene Therapy 11:1921-1931, 2000; and Klump etal., Gene Therapy 8:811-817, 2001).

Reporter Sequences

Any of the vectors provided herein can optionally include a sequenceencoding a reporter protein (“a reporter sequence”). Non-limitingexamples of reporter sequences are described herein. Additional examplesof reporter sequences are known in the art. In some embodiments, thereporter sequence can be used to verify the tissue-specific targetingcapabilities and tissue-specific promoter regulatory activity of any ofthe vectors described herein.

NTF3

Any of the vectors provided herein can optionally include a sequenceencoding a neurotrophin-3 (NTF3) protein. In some embodiments, a NTF3protein can have a sequence that is at least 70% identical, at least 72%identical, at least 74% identical, at least 76% identical, at least 78%identical, at least 80% identical, at least 82% identical, at least 84%identical, at least 86% identical, at least 88% identical, at least 90%identical, at least 92% identical, at least 94% identical, at least 96%identical, at least 98% identical, at least 99% identical, or 100%identical to SEQ ID NO: 78. In some embodiments, a NTF3 protein caninclude a sequence that is identical to SEQ ID NO: 78, except that itincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 amino acid substitutions and/or deletions.

In some embodiments, a NTF3 protein can be encoded by a sequence that isat least 70% identical, at least 72% identical, at least 74% identical,at least 76% identical, at least 78% identical, at least 80% identical,at least 82% identical, at least 84% identical, at least 86% identical,at least 88% identical, at least 90% identical, at least 92% identical,at least 94% identical, at least 96% identical, at least 98% identical,at least 99% identical, or 100% identical to SEQ ID NO: 79 or 80.

One skilled in the art would appreciate that mutation of amino acidsthat are not conserved between the same protein from different speciesis less likely to have an effect on the function of a protein andtherefore, these amino acids should be selected for mutation. Aminoacids that are conserved between the same protein from different speciesshould not be mutated, as these mutations are more likely to result in achange in the function of the protein. Non-limiting examples ofneutrophin-3 from other mammalian species are shown below.

Cow Neurotrophin-3 (SEQ ID NO: 81)

Rat Neurotrophin-3 (SEQ ID NO: 82)

Pig Neurotrophin-3 (SEQ ID NO: 83)

Flanking Regions Untranslated Regions (UTRs)

In some embodiments, any of the vectors described herein (e.g., any ofthe at least two different vectors) can include an untranslated region.In some embodiments, a vector can includes a 5′ UTR or a 3′ UTR.

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′ UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon. The3′ UTR starts immediately following the stop codon and continues untilthe transcriptional termination signal. There is growing body ofevidence about the regulatory roles played by the UTRs in terms ofstability of the nucleic acid molecule and translation. The regulatoryfeatures of a UTR can be incorporated into any of the vectors,compositions, kits, or methods as described herein to enhance thestability of an otoferlin protein.

Kozak Sequences

Natural 5′ UTRs include a sequence that plays a role in translationinitiation. They harbor signatures like Kozak sequences, which arecommonly known to be involved in the process by which the ribosomeinitiates translation of many genes. Kozak sequences have the consensussequence CCR(A/G)CCAUGG, where R is a purine (A or G) three basesupstream of the start codon (AUG), which is followed by another “G”. The5′ UTRs have also been known, e.g., to form secondary structures thatare involved in elongation factor binding.

For example, in some embodiments, a 5′ UTR is included in any of thevectors described herein. Non-limiting examples of 5′ UTRs includingthose from the following genes: albumin, serum amyloid A, ApolipoproteinA/B/E, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII,can be used to enhance expression of a nucleic acid molecule, such as amRNA.

In some embodiments, a 5′ UTR from a mRNA that is transcribed by a cellin the cochlea can be included in any of the vectors, compositions,kits, and methods described herein.

3′ UTRs are known to have stretches of adenosines and uridines embeddedin them. These AU-rich signatures are particularly prevalent in geneswith high rates of turnover. Based on their sequence features andfunctional properties, the AU-rich elements (AREs) can be separated intothree classes (Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen etal., Mol. Cell Biol. 15:2010-2018, 1995): Class I AREs contain severaldispersed copies of an AUUUA motif within U-rich regions. For example,c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two ormore overlapping UUAUUUA(U/A) (U/A) nonamers. GM-CSF and TNF-alpha mRNAsare examples that contain class II AREs. Class III AREs are less welldefined. These U-rich regions do not contain an AUUUA motif. Twowell-studied examples of this class are c-Jun and myogenin mRNAs.

Most proteins binding to the AREs are known to destabilize themessenger, whereas members of the ELAV family, most notably HuR, havebeen documented to increase the stability of mRNA. HuR binds to AREs ofall the three classes. Engineering the HuR specific binding sites intothe 3′ UTR of nucleic acid molecules will lead to HuR binding and thus,stabilization of the message in vivo.

In some embodiments, the introduction, removal, or modification of 3′UTR AREs can be used to modulate the stability of an mRNA encoding anotoferlin protein. In other embodiments, AREs can be removed or mutatedto increase the intracellular stability and thus increase translationand production of an otoferlin protein.

Splice Donor and Splice Acceptor Sequences

In other embodiments, non-UTR sequences may be incorporated into the 5′or 3′ UTRs. In some embodiments, introns or portions of intron sequencesmay be incorporated into the flanking regions of the polynucleotides inany of the vectors, compositions, kits, and methods provided herein.Incorporation of intronic sequences may increase protein production aswell as mRNA levels. An intron can be an intron from an otoferlin geneor can be an intron from a heterologous gene, e.g., a hybridadenovirus/mouse immunoglobulin intron (Yew et al., Human Gene Ter.8(5):575-584, 1997), an SV40 intron (Ostedgaard et al., Proc. Natl.Acad. Sci. U.S.A. 102(8):2952-2957, 2005), an MVM intron (Wu et al.,Mol. Ther. 16(2):280-289, 2008), a factor IX truncated intron 1 (Wu etal., Mol. Ther. 16(2):280-289, 2008; Kurachi et al., J. Biol. Chem.270(10):5276-5281, 1995), a chimeric J-globulin splicedonor/immunoglobulin heavy chain splice acceptor intron (Wu et al., Mol.Ther. 16(2):280-289, 2008; Choi et al., Mol. Brain 7:17, 2014), SV40late splice donor/splice acceptor intron (19S/16S) (Yew et al., HumanGene Ther. 8(5):575-584, 1997), hybrid adenovirus spice donor/IgG spliceacceptor (Choi et al., Mol. Brain 7:17, 1991; Huang and Gorman, Mol.Cell Biol. 10(4):1805-1810, 1990).

Non-limiting examples of a splice donor and splice acceptor sequencesare SEQ ID NOs: 64 and 65, respectively; SEQ ID NOs: 72 and 73,respectively; and SEQ ID NOs: 74 and 75, respectively. In someembodiments, the splice donor sequence has the sequence of SEQ ID NO:102. In some embodiments, a vector of construct of the presentdisclosure comprises a splice donor sequence of SEQ ID NO: 102. In somesuch embodiments, the vector or construct comprising a splice donorsequence (e.g., SEQ ID NO: 102) also comprises a 5′ portion of the OTOFgene or OTOF cDNA (e.g., SEQ ID NO: 101) upstream of the splice donorsequence. In some embodiments, the splice acceptor sequence has thesequence of SEQ ID NO: 106. In some embodiments, a vector or constructof the present disclosure comprises a splice acceptor sequence of SEQ IDNO: 106. In some such embodiments, the vector or construct comprising asplice acceptor sequence (e.g., SEQ ID NO: 106) also comprises a 3′portion of the OTOF gene or OTOF cDNA (e.g., SEQ ID NO: 107) downstreamof the splice acceptor sequence.

Destabilization Domains Any of the vectors provided herein canoptionally include a sequence encoding a destabilization domain (“adestabilization domain sequence”). A destabilization domain is an aminoacid sequence that decreases the in vivo or in vitro half-life of aprotein that includes the destabilization domain, e.g., as compared tothe same protein lacking the stabilization domain. For example, adestabilization domain may result in the targeting of a protein thatincludes the destabilization domain for proteosomal degradation.Non-limiting examples of destabilization domains include thedestabilizing domain of the E. coli dihydrofolate reductase (DHFR)(Iwamoto et al. (2010) Chem. Biol. 17(9): 981-998) and FK-506 bindingprotein (FKBP) (Wenlin et al. (2015) PLoS One 10(12): e0145783). SEQ IDNO: 53 is an exemplary amino acid sequence of a DHFR destabilizationdomain. Additional examples of destabilization domains are known in theart.

In some embodiments, any of the vectors provided herein can optionallyinclude a degradation sequence, e.g., a CL1 degradation sequence of SEQID NO: 71.

Recombinogenic Sequences

In some embodiments, one or more vectors or constructs of the presentdisclosure comprise(s) one or more recombinogenic sequences. In someembodiments, a recombinogenic sequence is or comprises a portion of agene sequence. In some embodiments, a recombinogenic sequence is derivedfrom an alkaline phosphatase gene. In some embodiments, a recombinogenicsequence is derived from an F1 phage. In some such embodiments, arecombinogenic sequence is an AK sequence derived from an F1 phage. Insome embodiments, such an AK recombinogenic sequence is SEQ ID NO: 103.In some embodiments of a dual vector system of the present disclosure,each of two vectors comprises a recombinogenic sequence.

In some embodiments, a composition of the present disclosure comprises afirst vector with a splice donor sequence (e.g., SEQ ID NO: 102) locateddownstream of a 5′ portion of the OTOF gene or OTOF cDNA (e.g., SEQ IDNO: 101) and upstream of an AK recombinogenic sequence (e.g., SEQ ID NO:103) and a second vector with a splice acceptor sequence (e.g., SEQ IDNO: 106) located upstream of a 3′ portion of the OTOF gene or OTOF cDNA(e.g., SEQ ID NO: 107) and downstream of an AK recombinogenic sequence(e.g., SEQ ID NO: 103).

Additional Sequences

Any of the vectors provided herein can optionally include additionalnucleotide sequences (“a stuffer sequence”) in order to optimize thetotal number of base pairs in the vector. For example, in order tooptimize packaging, each vector can be designed to contain a total ofabout 4,000 base pairs to about 4,700 base pairs, e.g., about 4,000 basepairs to about 4,650 base pairs, about 4,000 base pairs to about 4,600base pairs, about 4,000 base pairs to about 4,550 base pairs, about4,000 base pairs to about 4,500 base pairs, about 4,000 base pairs toabout 4,450 base pairs, about 4,000 base pairs to about 4,400 basepairs, about 4,000 base pairs to about 4,350 base pairs, about 4,000base pairs to about 4,300 base pairs, about 4,000 base pairs to about4,250 base pairs, about 4,000 base pairs to about 4,200 base pairs,about 4,000 base pairs to about 4,150 base pairs, about 4,000 base pairsto about 4,100 base pairs, about 4,000 base pairs to about 4,050 basepairs, about 4,050 base pairs to about 4,700 base pairs, about 4,050base pairs to about 4,650 base pairs, about 4,050 base pairs to about4,600 base pairs, about 4,050 base pairs to about 4,550 base pairs,about 4,050 base pairs to about 4,500 base pairs, about 4,050 base pairsto about 4,450 base pairs, about 4,050 base pairs to about 4,400 basepairs, about 4,050 base pairs to about 4,350 base pairs, about 4,050base pairs to about 4,300 base pairs, about 4,050 base pairs to about4,250 base pairs, about 4,050 base pairs to about 4,200 base pairs,about 4,050 base pairs to about 4,150 base pairs, about 4,050 base pairsto about 4,100 base pairs, about 4,100 base pairs to about 4,700 basepairs, about 4,100 base pairs to about 4,650 base pairs, about 4,100base pairs to about 4,600 base pairs, about 4,100 base pairs to about4,550 base pairs, about 4,100 base pairs to about 4,500 base pairs,about 4,100 base pairs to about 4,450 base pairs, about 4,100 base pairsto about 4,400 base pairs, about 4,100 base pairs to about 4,350 basepairs, about 4,100 base pairs to about 4,300 base pairs, about 4,100base pairs to about 4,250 base pairs, about 4,100 base pairs to about4,200 base pairs, about 4,100 base pairs to about 4,150 base pairs,about 4,150 base pairs to about 4,700 base pairs, about 4,150 base pairsto about 4,650 base pairs, about 4,150 base pairs to about 4,600 basepairs, about 4,150 base pairs to about 4,550 base pairs, about 4,150base pairs to about 4,500 base pairs, about 4,150 base pairs to about4,450 base pairs, about 4,150 base pairs to about 4,400 base pairs,about 4,150 base pairs to about 4,350 base pairs, about 4,150 base pairsto about 4,300 base pairs, about 4,150 base pairs to about 4,250 basepairs, about 4,150 base pairs to about 4,200 base pairs, about 4,200base pairs to about 4,700 base pairs, about 4,200 base pairs to about4,650 base pairs, about 4,200 base pairs to about 4,600 base pairs,about 4,200 base pairs to about 4,550 base pairs, about 4,200 base pairsto about 4,500 base pairs, about 4,200 base pairs to about 4,450 basepairs, about 4,200 base pairs to about 4,400 base pairs, about 4,200base pairs to about 4,350 base pairs, about 4,200 base pairs to about4,300 base pairs, about 4,200 base pairs to about 4,250 base pairs,about 4,250 base pairs to about 4,700 base pairs, about 4,250 base pairsto about 4,650 base pairs, about 4,250 base pairs to about 4,600 basepairs, about 4,250 base pairs to about 4,550 base pairs, about 4,250base pairs to about 4,500 base pairs, about 4,250 base pairs to about4,450 base pairs, about 4,250 base pairs to about 4,400 base pairs,about 4,250 base pairs to about 4,350 base pairs, about 4,250 base pairsto about 4,300 base pairs, about 4,300 base pairs to about 4,700 basepairs, about 4,300 base pairs to about 4,650 base pairs, about 4,300base pairs to about 4,600 base pairs, about 4,300 base pairs to about4,550 base pairs, about 4,300 base pairs to about 4,500 base pairs,about 4,300 base pairs to about 4,450 base pairs, about 4,300 base pairsto about 4,400 base pairs, about 4,300 base pairs to about 4,350 basepairs, about 4,350 base pairs to about 4,700 base pairs, about 4,350base pairs to about 4,650 base pairs, about 4,350 base pairs to about4,600 base pairs, about 4,350 base pairs to about 4,550 base pairs,about 4,350 base pairs to about 4,500 base pairs, about 4,350 base pairsto about 4,450 base pairs, about 4,350 base pairs to about 4,400 basepairs, about 4,400 base pairs to about 4,700 base pairs, about 4,400base pairs to about 4,650 base pairs, about 4,400 base pairs to about4,600 base pairs, about 4,400 base pairs to about 4,550 base pairs,about 4,400 base pairs to about 4,500 base pairs, about 4,400 base pairsto about 4,450 base pairs, about 4,450 base pairs to about 4,700 basepairs, about 4,450 base pairs to about 4,650 base pairs, about 4,450base pairs to about 4,600 base pairs, about 4,450 base pairs to about4,550 base pairs, about 4,450 base pairs to about 4,500 base pairs,about 4,500 base pairs to about 4,700 base pairs, about 4,500 base pairsto about 4,650 base pairs, about 4,500 base pairs to about 4,600 basepairs, about 4,500 base pairs to about 4,550 base pairs, about 4,550base pairs to about 4,700 base pairs, about 4,550 base pairs to about4,650 base pairs, about 4,550 base pairs to about 4,600 base pairs,about 4,600 base pairs to about 4,700 base pairs, about 4,600 base pairsto about 4,650 base pairs, or about 4,650 base pairs to about 4,700 basepairs (inclusive).

A stuffer sequence can be any nucleotide sequence, e.g., up to 1000 bp,that can be included in any of the vectors described herein that is nottranscribed and that does not serve a regulatory function in order toachieve a desirable vector size (e.g., a vector size of about 4 kb toabout 5 kb, or any of the vector sizes provided herein). For example, astuffer sequence can be any nucleotide sequence of about 100 bp to about1000 bp (e.g., about 100 bp to about 900 bp, about 100 bp to about 800bp, about 100 bp to about 700 bp, about 100 bp to about 600 bp, about100 bp to about 500 bp, about 100 bp to about 400 bp, about 100 bp toabout 300 bp, about 100 bp to about 100 bp, about 200 bp to about 1000bp, about 200 bp to about 900 bp, about 200 bp to about 800 bp, about200 bp to about 700 bp, about 200 bp to about 600 bp, about 200 bp toabout 500 bp, about 200 bp to about 400 bp, about 200 bp to about 300bp, about 300 bp to about 1000 bp, about 300 bp to about 900 bp, about300 bp to about 800 bp, about 300 bp to about 700 bp, about 300 bp toabout 600 bp, about 300 bp to about 500 bp, about 300 bp to about 400bp, about 400 bp to about 1000 bp, about 400 bp to about 900 bp, about400 bp to about 800 bp, about 400 bp to about 700 bp, about 400 bp toabout 600 bp, about 400 bp to about 500 bp, about 500 bp to about 1000bp, about 500 bp to about 900 bp, about 500 bp to about 800 bp, about500 bp to about 700 bp, about 500 bp to about 600 bp, about 600 bp toabout 1000 bp, about 600 bp to about 900 bp, about 600 bp to about 800bp, about 600 bp to about 700 bp, about 700 bp to about 1000 bp, about700 bp to about 900 bp, about 700 bp to about 800 bp, about 800 bp toabout 1000 bp, about 800 bp to about 900 bp, about 900 bp to about 1000bp, about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 550bp, about 600 bp, about 650 bp, about 700 bp, about 750 bp, about 800bp, about 850 bp, about 900 bp, about 950 bp, or about 1000 bp. SEQ IDNOs. 54-58, 90 and 91 are exemplary human factor VIII stuffer sequencesthat can be used in any of the vectors described herein. Additionalstuffer sequences are known in the art. Exemplary vectors that includestuffer sequences are shown in FIGS. 21-31, 36, 37, 59-63 and 66 .

Dual AAV Vector Compositions

In some embodiments, the present disclosure provides compositionscomprising one or more vectors to deliver a therapeutic gene, e.g., anentire therapeutic gene or a functional portion thereof, to a subject inneed thereof. For example, in some embodiments, the otoferlin gene istoo large to be packaged into a single recombinant vector, e.g., arecombinant AAV vector. Accordingly, in some embodiments, two or morevectors are employed to deliver a therapeutic gene, e.g., an entiretherapeutic gene to a subject in need thereof. For example, in someembodiments, a dual vector system is used, wherein each of two vectorscomprises a portion of the human otoferlin gene and, when delivered invivo, the constructs come together to generate a polynucleotide thatencodes a full length, functional, otoferlin protein. In someembodiments, one or more strategies is used, for example, (i) aconcatemerization-trans-splicing strategy, (ii) a hybridintronic-homologous recombination-trans-splicing strategy, and (iii) anexonic homologous recombination strategy, as summarized by Pryadkina etal. Meth Clin Devel 2015, 2:15009.

Mammalian Cells

Also provided herein is a cell (e.g., a mammalian cell) that includesany of the nucleic acids, vectors (e.g., at least two different vectorsdescribed herein), or compositions described herein. Skilledpractitioners will appreciate that the nucleic acids and vectorsdescribed herein can be introduced into any mammalian cell. Non-limitingexamples of vectors and methods for introducing vectors into mammaliancells are described herein. In some embodiments, the cell is a humancell, a mouse cell, a porcine cell, a rabbit cell, a dog cell, a catcell, a rat cell, a sheep cell, a cat cell, a horse cell, or a non-humanprimate cell. In some embodiments, the cell is a specialized cell of thecochlea. In some embodiments, the cell is a cochlear inner hair cell ora cochlear outer hair cell. In some embodiments, the cell is a cochlearinner hair cell. In some embodiments, the cell is a cochlear inner haircell.

In some embodiments, the mammalian cell is in vitro. In someembodiments, the mammalian cell is present in a mammal. In someembodiments, the mammalian cell is obtained from a subject. In someembodiments, the mammalian cell is an autologous cell obtained from asubject and/or is cultured ex vivo.

Methods of Use

Method of Introduction into Cochlea

Also provided herein is a method of introducing into a cochlea of amammal (e.g., a human) a therapeutically effective amount of any of thecompositions described herein. Also provided are methods of increasingexpression of an active otoferlin protein (e.g., a full-length otoferlinprotein) in an inner hair cell in a cochlea of a mammal (e.g., a human)that include introducing into the cochlea of the mammal atherapeutically effective amount of any of the compositions describedherein. Also provided are methods of treating non-symptomaticsensorineural hearing loss in a subject (e.g., a human) identified ashaving a defective otoferlin gene, wherein the methods includeadministering a therapeutically effective amount of any of thecompositions described herein into a cochlea of a subject. In someexamples, the methods described herein can further include administeringa neurotrophic factor to a cochlea of a subject (e.g., at substantiallythe same time as or before, or after, any of the compositions describedherein are administered to the subject). In some examples, the methodsdescribed herein can further include administering a cochlear implant toa subject (e.g., at substantially the same time as or before, or after,any of the compositions described herein are administered to thesubject).

In some embodiments of any of these methods, the mammal has beenpreviously identified as having a defective otoferlin gene (e.g., anotoferlin gene having a mutation that results in a decrease in theexpression and/or activity of an otoferlin protein encoded by the gene).Some embodiments of any of these methods further include, prior to theintroducing or administering step, determining that the subject has adefective otoferlin gene. Some embodiments of any of these methods canfurther include detecting a mutation in an otoferlin gene in a subject.Some embodiments of any of the methods can further include identifyingor diagnosing a subject as having non-symptomatic sensorineural hearingloss.

In some embodiments of any of these methods, two or more doses of any ofthe compositions described herein are introduced or administered intothe cochlea of the mammal or subject. Some embodiments of any of thesemethods can include introducing or administering a first dose of thecomposition into the cochlea of the mammal or subject, assessing hearingfunction of the mammal or subject following the introducing or theadministering of the first dose, and administering an additional dose ofthe composition into the cochlea of the mammal or subject found not tohave a hearing function within a normal range (e.g., as determined usingany test for hearing known in the art).

In some embodiments of any of the methods described herein, thecomposition can be formulated for intra-cochlear administration. In someembodiments of any of the methods described herein, the compositionsdescribed herein can be administered via intra-cochlear administrationor local administration. In some embodiments of any of the methodsdescribed herein, the compositions are administered through the use of amedical device (e.g., any of the exemplary medical devices describedherein).

In some embodiments, intra-cochlear administration can be performedusing any of the methods described herein or known in the art. Forexample, a composition can be administered or introduced into thecochlea using the following surgical technique: first usingvisualization with a 0 degree, 2.5-mm rigid endoscope, the externalauditory canal is cleared and a round knife is used to sharply delineatean approximately 5-mm tympanomeatal flap. The tympanomeatal flap is thenelevated and the middle ear is entered posteriorly. The chorda tympaninerve is identified and divided, and a currette is used to remove thescutal bone, exposing the round window membrane. To enhance apicaldistribution of the administered or introduced composition, a surgicallaser may be used to make a small 2-mm fenestration in the oval windowto allow for perilymph displacement during trans-round window membraneinfusion of the composition. The microinfusion device is then primed andbrought into the surgical field. The device is maneuvered to the roundwindow, and the tip is seated within the bony round window overhang toallow for penetration of the membrane by the microneedle(s). Thefootpedal is engaged to allow for a measured, steady infusion of thecomposition. The device is then withdrawn and the round window andstapes foot plate are sealed with a gelfoam patch.

In some embodiments, the present disclosure describes a deliveryapproach that utilizes a minimally invasive, well-accepted surgicaltechnique for accessing the middle ear and/or inner ear through theexternal auditory canal. The procedure includes opening one of thephysical barriers between the middle and inner ear at the oval window,and subsequently using a device disclosed herein, e.g., as shown inFIGS. 82-87 (or microcatheter) to deliver a composition disclosed hereinat a controlled flow rate and in a fixed volume, via the round windowmembrane.

In some embodiments, surgical procedures for mammals (e.g., rodents(e.g., mice, rats, hamsters, or rabbits), primates (e.g., NHP (e.g.,macaque, chimpanzees, monkeys, or apes) or humans) may include ventingto increase AAV vector transduction rates along the length of thecochlea. In some embodiments, absence of venting during surgery mayresult in lower AAV vector cochlear cell transduction rates whencompared to AAV vector cochlear cell transduction rates followingsurgeries performed with venting. In some embodiments, ventingfacilitates transduction rates of about 75-100% of IHCs throughout thecochlea. In some embodiments, venting permits IHC transduction rates ofabout 50-70%, about 60-80%, about 70-90%, or about 80-100% at the baseof the cochlea. In some embodiments, venting permits IHC transductionrates of about 50-70%, about 60-80%, about 70-90%, or about 80-100% atthe apex of the cochlea.

A delivery device described herein may be placed in a sterile field ofan operating room and the end of a tubing may be removed from thesterile field and connected to a syringe that has been loaded with acomposition disclosed herein (e.g., one or more AAV vectors) and mountedin the pump. After appropriate priming of the system in order to removeany air, a needle may then be passed through the middle ear undervisualization (surgical microscope, endoscope, and/or distal tipcamera). A needle (or microneedle) may be used to puncture the RWM. Theneedle may be inserted until a stopper contacts the RWM. The device maythen be held in that position while a composition disclosed herein isdelivered at a controlled flow rate to the inner ear, for a selectedduration of time. In some embodiments, the flow rate (or infusion rate)may include a rate of about 30 μL/min, or from about 25 μL/min to about35 μL/min, or from about 20 μL/min to about 40 μL/min, or from about 20μL/min to about 70 μL/min, or from about 20 μL/min to about 90 μL/min,or from about 20 μL/min to about 100 μL/min. In some embodiments, theflow rate is about 20 μL/min, about 30 μL/min, about 40 μL/min, about 50μL/min, about 60 μL/min, about 70 μL/min, about 80 μL/min, about 90μL/min or about 100 μL/min. In some embodiments, the selected durationof time (that is, the time during which a composition disclosed hereinis flowing) may be about 3 minutes, or from about 2.5 minutes to about3.5 minutes, or from about 2 minutes to about 4 minutes, or from about1.5 minutes to about 4.5 minutes, or from about 1 minute to about 5minutes. In some embodiments, the total volume of a compositiondisclosed herein that flows to the inner ear may be about 0.09 mL, orfrom about 0.08 mL to about 0.10 mL, or from about 0.07 mL to about 0.11mL. In some embodiments, the total volume of a composition disclosedherein equates to from about 40% to about 50% of the volume of the innerear.

Once the delivery has been completed, the device may be removed. In someembodiments, a device described herein, may be configured as asingle-use disposable product. In other embodiments, a device describedherein may be configured as a multi-use, sterilizable product, forexample, with a replaceable and/or sterilizable needle sub-assembly.Single use devices may be appropriately discarded (for example, in abiohazard sharps container) after administration is complete.

In some embodiments, a composition disclosed herein comprises one or aplurality of AAV vectors. In some embodiments, when more than one AAVvector is included in the composition, the AAV vectors are eachdifferent. In some embodiments, an AAV vector comprises an OTOF codingregion, e.g., as described herein. In some embodiments, a compositioncomprises an rAAV particle comprising an AAV vector described herein. Insome embodiments, the r AAV particle is encapsidated by an Anc80 capsid.In some embodiment, the Anc80 capsid comprises a polypeptide of SEQ IDNO: 109.

Subjects

In some embodiments of any of the methods described herein, the subjector mammal is a rodent, a non-human primate, or a human. In someembodiments of any of the methods described herein, the subject ormammal is an adult, a teenager, a juvenile, a child, a toddler, aninfant, or a newborn. In some embodiments of any of the methodsdescribed herein, the subject or mammal is 1-5, 1-10, 1-20, 1-30, 1-40,1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-110, 2-5, 2-10, 10-20, 20-30,30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 10-30, 10-40,10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-110, 20-40, 20-50, 20-60,20-70, 20-80, 20-90, 20-100, 20-110, 30-50, 30-60, 30-70, 30-80, 30-90,30-100, 40-60, 40-70, 40-80, 40-90, 40-100, 50-70, 50-80, 50-90, 50-100,60-80, 60-90, 60-100, 70-90, 70-100, 70-110, 80-100, 80-110, or 90-110years of age. In some embodiments of any of the methods describedherein, the subject or mammal is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11months of age.

In some embodiments of any of the methods described herein, the methodsresult in improvement in hearing (e.g., any of the metrics fordetermining improvement in hearing described herein) in a subject inneed thereof for at least 10 days, at least 15 days, at least 20 days,at least 25 days, at least 30 days, at least 35 days, at least 40 days,at least 45 days, at least 50 days, at least 55 days, at least 60 days,at least 65 days, at least 70 days, at least 75 days, at least 80 days,at least 85 days, at least 100 days, at least 105 days, at least 110days, at least 115 days, at least 120 days, at least 5 months, at least6 months, at least 7 months, at least 8 months, at least 9 months, atleast 10 months, at least 11 months, or at least 12 months.

In some embodiments of any of the methods described herein, the subjector mammal has or is at risk of developing non-syndromic sensorineuralhearing loss. In some embodiments of any of the methods describedherein, the subject or mammal has been previously identified as having amutation in an otoferlin gene. In some embodiments of any of the methodsdescribed herein, the subject or mammal has any of the mutations in anotoferlin gene that are described herein or are known in the art to beassociated with non-symptomatic sensorineural hearing loss.

In some embodiments of any of the methods described herein, the subjector mammal has been identified as being a carrier of a mutation in anotoferlin gene (e.g., via genetic testing). In some embodiments of anyof the methods described herein, the subject or human has beenidentified as having a mutation in an otoferlin gene and has beendiagnosed with non-symptomatic sensorineural hearing loss. In someembodiments of any of the methods described herein, the subject or humanhas been identified as having non-symptomatic sensorineural hearingloss.

In some embodiments, successful treatment of non-symptomaticsensorineural hearing loss can be determined in a subject using any ofthe conventional functional hearing tests known in the art. Non-limitingexamples of functional hearing tests are various types of audiometricassays (e.g., pure-tone testing, speech testing, test of the middle ear,auditory brainstem response, and otoacoustic emissions).

Increasing Expression of Active Otoferlin

Also provided herein are methods of increasing expression of an activeotoferlin protein (e.g., a full-length otoferlin protein) in a mammaliancell that include introducing any of the compositions described hereininto a mammalian cell. In some embodiments of these methods, a mammaliancell is a cochlear inner hair cell. In some embodiments of thesemethods, a mammalian cell is a human cell (e.g., a human cochlear innerhair cell). In some embodiments of these methods, a mammalian cell is invitro. In some embodiments of these methods, a mammalian cell is in amammal. In some embodiments of these methods, a mammalian cell isoriginally obtained from a mammal and/or is cultured ex vivo. In someembodiments, a mammalian cell has previously been determined to have adefective otoferlin gene.

Methods for introducing any of the compositions described herein into amammalian cell are known in the art (e.g., via lipofection or throughthe use of a viral vector, e.g., any of the viral vectors describedherein).

In some embodiment, an increase in expression of an active otoferlinprotein (e.g., a full-length otoferlin protein) as described herein is,e.g., as compared to a control or to the level of expression of anactive otoferlin protein (e.g., a full-length otoferlin protein) priorto the introduction of the vector(s).

Methods of Detecting Otoferlin

Methods of detecting expression and/or activity of otoferlin are knownin the art. In some embodiments, the level of expression of an otoferlinprotein can be detected directly (e.g., detecting otoferlin protein ordetecting otoferlin mRNA). Non-limiting examples of techniques that canbe used to detect expression and/or activity of otoferlin directlyinclude: real-time PCR, Western blotting, immunoprecipitation,immunohistochemistry, or immunofluorescence. In some embodiments,expression of an otoferlin protein can be detected indirectly (e.g.,through functional hearing tests).

Compositions

Among other things, the present disclosure provides compositions. Insome embodiments, a composition comprises a construct as describedherein. In some embodiments, a composition comprises one or moreconstructs as described herein. In some embodiments, a compositioncomprises a plurality of constructs as described herein. In someembodiments, when more than one construct is included in thecomposition, the constructs are each different.

In some embodiments, a composition comprises an AAV vector as describedherein. In some embodiments, a composition comprises one or more AAVvectors as described herein. In some embodiments, a compositioncomprises a plurality of AAV vectors. In some embodiments, when morethan one AAV vector is included in the composition, the AAV vectors areeach different. In some embodiments, an AAV vector comprises an OTOFcoding region, e.g., as described herein.

In some embodiments, a composition comprises one or more recombinant AAV(rAAV) particles. In some embodiments, an rAAV particle comprises arecombinant AAV vector (rAAV). In some embodiments, an rAAV particle isencapsidated by an Anc80 capsid. In some embodiments, the Anc80 capsidcomprises a polypeptide of SEQ ID NO: 109.

In some embodiments, a composition is or comprises a pharmaceuticalcomposition.

In some embodiments, a composition described herein is in a solution.

Dosing and Volume of Administration

In some embodiments, a composition disclosed herein, e.g., one or aplurality of AAV vectors disclosed herein, is administered as a singledose or as a plurality of doses.

In some embodiments, a composition disclosed herein is administered as asingle dose. In some embodiments, a composition disclosed herein isadministered as a plurality of doses, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10doses.

In some embodiments, a composition disclosed herein (e.g., a compositioncomprising one or a plurality of AAV vectors disclosed herein) isadministered at a volume of about 0.01 mL, about 0.02 mL, about 0.03 mL,about 0.04 mL, about 0.05 mL, about 0.06 mL, about 0.07 mL, about 0.08mL, about 0.09 mL, about 1.00 mL, about 1.10 mL, about 1.20 mL, about1.30 mL, about 1.40 mL, about 1.50 mL, about 1.60 mL, about 1.70 mL,about 1.80 mL, about 1.90 mL, or about 2.00 mL. In some embodiments, acomposition disclosed herein is administered at a volume of about 0.01mL. In some embodiments, a composition disclosed herein is administeredat a volume of about 0.02 mL. In some embodiments, a compositiondisclosed herein is administered at a volume of about 0.03 mL. In someembodiments, a composition disclosed herein is administered at a volumeof about 0.04 mL. In some embodiments, a composition disclosed herein isadministered at a volume of about 0.05 mL. In some embodiments, acomposition disclosed herein is administered at a volume of about 0.06mL. In some embodiments, a composition disclosed herein is administeredat a volume of about 0.07 mL. In some embodiments, a compositiondisclosed herein is administered at a volume of about 0.08 mL. In someembodiments, a composition disclosed herein is administered at a volumeof about 0.09 mL. In some embodiments, a composition disclosed herein isadministered at a volume of about 1.00 mL. In some embodiments, acomposition disclosed herein is administered at a volume of about 1.10mL. In some embodiments, a composition disclosed herein is administeredat a volume of about 1.20 mL. In some embodiments, a compositiondisclosed herein is administered at a volume of about 1.30 mL. In someembodiments, a composition disclosed herein is administered at a volumeof about 1.40 mL. In some embodiments, a composition disclosed herein isadministered at a volume of about 1.50 mL. In some embodiments, acomposition disclosed herein is administered at a volume of about 1.60mL. In some embodiments, a composition disclosed herein is administeredat a volume of about 1.70 mL. In some embodiments, a compositiondisclosed herein is administered at a volume of about 1.80 mL. In someembodiments, a composition disclosed herein is administered at a volumeof about 1.90 mL. In some embodiments, a composition disclosed herein isadministered at a volume of about 2.00 mL.

In some embodiments, a composition disclosed herein (e.g., a compositioncomprising one or a plurality of AAV vectors disclosed herein) isadministered at a volume of about 0.01 to 2.00 mL, about 0.02 to 1.90mL, about 0.03 to 1.8 mL, about 0.04 to 1.70 mL, about 0.05 to 1.60 mL,about 0.06 to 1.50 mL, about 0.06 to 1.40 mL, about 0.07 to 1.30 mL,about 0.08 to 1.20 mL, or about 0.09 to 1.10 mL. In some embodiments acomposition disclosed herein (e.g., a composition comprising one or aplurality of AAV vectors disclosed herein) is administered at a volumeof about 0.01 to 2.00 mL, about 0.02 to 2.00 mL, about 0.03 to 2.00 mL,about 0.04 to 2.00 mL, about 0.05 to 2.00 mL, about 0.06 to 2.00 mL,about 0.07 to 2.00 mL, about 0.08 to 2.00 mL, about 0.09 to 2.00 mL,about 0.01 to 1.90 mL, about 0.01 to 1.80 mL, about 0.01 to 1.70 mL,about 0.01 to 1.60 mL, about 0.01 to 1.50 mL, about 0.01 to 1.40 mL,about 0.01 to 1.30 mL, about 0.01 to 1.20 mL, about 0.01 to 1.10 mL,about 0.01 to 1.00 mL, about 0.01 to 0.09 mL.

Pharmaceutical Compositions and Kits

Pharmaceutical compositions of the present disclosure may comprise anucleic acid, e.g., one or a plurality of AAV vectors, as describedherein, in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. In someembodiments, a pharmaceutical composition may comprise one or more AAVvectors, e.g., one or more AAV constructs encapsidated by one or moreAAV serotype capsids, as described herein. In some embodiments, apharmaceutical composition may comprise buffers such as neutral bufferedsaline, phosphate buffered saline and the like; carbohydrates such asglucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptidesor amino acids such as glycine; antioxidants; chelating agents such asEDTA or glutathione; adjuvants (e.g., aluminum hydroxide); andpreservatives. Compositions of the present disclosure are in one aspectformulated for intra-cochlear administration. Compositions of thepresent disclosure are in one aspect formulated for intravenousadministration.

In some embodiments, the therapeutic compositions are formulated forintra-cochlear administration. In some embodiments, the therapeuticcompositions are formulated to comprise a lipid nanoparticle. In someembodiments, the therapeutic compositions are formulated to comprise apolymeric nanoparticle. In some embodiments, the therapeuticcompositions are formulated to comprise a mini-circle DNA. In someembodiments, the therapeutic compositions are formulated to comprise aCELiD DNA. In some embodiments, the therapeutic compositions areformulated to comprise a synthetic perilymph solution. An exemplarysynthetic perilymph solution includes 20-200 mM NaCl; 1-5 mM KCl; 0.1-10mM CaCl₂); 1-10 mM glucose; and 2-50 mM HEPES, with a pH between about 6and about 9.

In some embodiments, any of the compositions described herein canfurther include one or more agents that promote the entry of a nucleicacid or any of the vectors described herein into a mammalian cell (e.g.,a liposome or cationic lipid). In some embodiments, any of the vectorsdescribed herein can be formulated using natural and/or syntheticpolymers. Non-limiting examples of polymers that may be included in anyof the compositions described herein can include, but are not limitedto, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.),formulations from Mirus Bio (Madison, Wis.) and Roche Madison (Madison,Wis.), PhaseRX polymer formulations such as, without limitation, SMARTTPOLYMER TECHNOLOGY® (PhaseRX, Seattle, Wash.), DMRI/DOPE, poloxamer,VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly (lactic-co-glycolic acid) (PLGA) polymers, RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.), and pH responsive co-blockpolymers, such as, but not limited to, those produced by PhaseRX(Seattle, Wash.). Many of these polymers have demonstrated efficacy indelivering oligonucleotides in vivo into a mammalian cell (see, e.g.,deFougerolles, Human Gene Ther. 19:125-132, 2008; Rozema et al., Proc.Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Rozema et al., Proc.Natl. Acad. Sci. U.S.A. 104:12982-12887, 2007; Hu-Lieskovan et al.,Cancer Res. 65:8984-8982, 2005; Heidel et al., Proc. Natl. Acad. Sci.U.S.A. 104:5715-5721, 2007). Any of the compositions described hereincan be, e.g., a pharmaceutical composition. In some embodiments, thecomposition includes a pharmaceutically acceptable carrier (e.g.,phosphate buffered saline, saline, or bacteriostatic water). Uponformulation, solutions will be administered in a manner compatible withthe dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, injectable gels, drug-releasecapsules, and the like.

As used herein, the term “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial agents, antifungalagents, and the like that are compatible with pharmaceuticaladministration. Supplementary active compounds can also be incorporatedinto any of the compositions described herein.

In some embodiments, a single dose of any of the compositions describedherein can include a total sum amount of the at least two differentvectors of at least 1 ng, at least 2 ng, at least 4 ng, about 6 ng,about 8 ng, at least 10 ng, at least 20 ng, at least 30 ng, at least 40ng, at least 50 ng, at least 60 ng, at least 70 ng, at least 80 ng, atleast 90 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least400 ng, at least 500 ng, at least 1 μg, at least 2 μg, at least 4 μg, atleast 6 μg, at least 8 μg, at least 10 μg, at least 12 μg, at least 14μg, at least 16 g, at least 18 ag, at least 20 ag, at least 22 ag, atleast 24 μg, at least 26 μg, at least 28 μg, at least 30 μg at least 32μg, at least 34 μg, at least 36 μg, at least 38 μg, at least 40 g, atleast 42 μg, at least 44 μg, at least 46 μg, at least 48 μg, at least 50μg, at least 52 g, at least 54 μg, at least 56 μg, at least 58 μg, atleast 60 μg, at least 62 μg, at least 64 μg, at least 66 μg, at least 68μg, at least 70 μg, at least 72 μg, at least 74 μg, at least 76 g, atleast 78 μg, at least 80 μg, at least 82 μg, at least 84 μg, at least 86μg, at least 88 g, at least 90 μg, at least 92 μg, at least 94 μg, atleast 96 μg, at least 98 μg, at least 100 μg, at least 102 μg, at least104 μg, at least 106 μg, at least 108 μg, at least 110 μg, at least 112μg, at least 114 μg, at least 116 μg, at least 118 μg, at least 120 μg,at least 122 μg, at least 124 μg, at least 126 μg, at least 128 μg, atleast 130 μg at least 132 μg, at least 134 μg, at least 136 μg, at least138 μg, at least 140 μg, at least 142 μg, at least 144 μg, at least 146μg, at least 148 μg, at least 150 μg, at least 152 μg, at least 154 μg,at least 156 μg, at least 158 μg, at least 160 μg, at least 162 μg, atleast 164 μg, at least 166 μg, at least 168 μg, at least 170 μg, atleast 172 μg, at least 174 μg, at least 176 μg, at least 178 μg, atleast 180 μg, at least 182 μg, at least 184 μg, at least 186 μg, atleast 188 μg, at least 190 μg, at least 192 μg, at least 194 μg, atleast 196 μg, at least 198 μg, or at least 200 μg, e.g., in a bufferedsolution.

The compositions provided herein can be, e.g., formulated to becompatible with their intended route of administration. A non-limitingexample of an intended route of administration is local administration(e.g., intra-cochlear administration).

In some embodiments, the therapeutic compositions are formulated toinclude a lipid nanoparticle. In some embodiments, the therapeuticcompositions are formulated to include a polymeric nanoparticle. In someembodiments, the therapeutic compositions are formulated to comprise amini-circle DNA. In some embodiments, the therapeutic compositions areformulated to comprise a CELiD DNA. In some embodiments, the therapeuticcompositions are formulated to comprise a synthetic perilymph solution.An exemplary synthetic perilymph solution includes 20-200 mM NaCl; 1-5mM KCl; 0.1-10 mM CaCl₂; 1-10 mM glucose; 2-50 mM HEPES, having a pH ofbetween about 6 and about 9.

Also provided are kits including any of the compositions describedherein. In some embodiments, a kit can include a solid composition(e.g., a lyophilized composition including the at least two differentvectors described herein) and a liquid for solubilizing the lyophilizedcomposition. In some embodiments, a kit can include a pre-loaded syringeincluding any of the compositions described herein.

In some embodiments, a kit includes a vial comprising any of thecompositions described herein (e.g., formulated as an aqueouscomposition, e.g., an aqueous pharmaceutical composition).

In some embodiments, a kit can include instructions for performing anyof the methods described herein.

Routes of Administration

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present disclosure intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trials examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MHLVs). MLVs generally havediameters of from 25 nm to 4 Tm. Sonication of MHLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500.ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 Tm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (i.e., ultrasound) hasbeen used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a subject transarterially, subcutaneously, intradermally, intranodally, intramedullary,intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.In one aspect, the nucleic acid compositions of the present disclosureare administered to a subject by intradermal or subcutaneous injection.In one aspect, the nucleic compositions of the present disclosure areadministered by i.v. injection.

Devices and Surgical Methods

Provided herein are technologies (e.g., systems, methods, devices, etc.)that may be used, in some embodiments, for treating deafness and otherhearing-associated diseases, disorders and conditions. Examples of suchtechnologies are also included in, e.g., WO2017223193 and WO2019084145,each of which is herein incorporated by reference in its entirety. Inone aspect, the present disclosure provides therapeutic delivery systemsfor treating deafness and other hearing-associated diseases, disordersand conditions. In one aspect, provided are therapeutic delivery systemsthat include i) a medical device capable of creating one or a pluralityof incisions in a round window membrane of an inner ear of a humansubject in need thereof, and ii) an effective dose of a therapeuticcomposition comprising one or a plurality of adeno-associated viral(AAV) vectors, wherein the one or the plurality of AAV vectors arecapable of constituting a full-length auditory polypeptide messenger RNAin a target cell of the inner ear. In some embodiments, of a means forperforming a surgical method, the method comprises the steps of:administering intra-cochlearly to a human subject in need thereof aneffective dose of a therapeutic composition of the present disclosure,wherein the therapeutic composition is capable of being administered byusing a medical device which comprises: a) a means for creating one or aplurality of incisions in a round window membrane; and b) an effectivedose of a therapeutic composition.

Provided herein are surgical methods for treatment of hearing loss. Inone aspect, the methods include the steps of: introducing into a cochleaof a human subject a first incision at a first incision point; andadministering intra-cochlearly an effective dose of a therapeuticcomposition (e.g., any of the compositions described herein) as providedherein. In one embodiment, a therapeutic composition (e.g., any of thecompositions described herein) is administered to the subject at a firstincision point. In one embodiment, a therapeutic composition isadministered to a subject into or through a first incision. In oneembodiment, a therapeutic composition is administered to a subject intoor through a cochlea oval window membrane. In one embodiment, atherapeutic composition is administered to a subject into or through acochlea round window membrane.

In some embodiments, a composition disclosed herein can be administeredto a subject with a surgical procedure. In some embodiments,administration, e.g., via a surgical procedure, comprises injecting acomposition disclosed herein via a delivery device as described hereininto the inner ear. In some embodiments, a surgical procedure disclosedherein comprises performing a transcanal tympanotomy; performing alaser-assisted micro-stapedotomy; and injecting a composition disclosedherein via a delivery device as described herein into the inner ear.

In some embodiments, a surgical procedure comprises performing atranscanal tympanotomy; performing a laser-assisted micro-stapedotomy;injecting a composition disclosed herein via a delivery device asdescribed herein into the inner ear; applying sealant around the roundwindow and/or an oval window of the subject; and lowering atympanomeatal flap of the subject to the anatomical position.

In some embodiments, a surgical procedure comprises performing atranscanal tympanotomy; preparing a round window of the subject;performing a laser-assisted micro-stapedotomy; preparing both a deliverydevice as described herein and a composition disclosed herein fordelivery to the inner ear; injecting a composition disclosed herein viathe delivery device into the inner ear; applying sealant around theround window and/or an oval window of the subject; and lowering atympanomeatal flap of the subject to the anatomical position.

In some embodiments, performing a laser-assisted micro-stapedotomyincludes using a KTP otologic laser and/or a C02 otologic laser.

In some embodiments, a composition comprises one or a plurality of AAVvectors. In some embodiments, when more than one AAV vector is includedin the composition, the AAV vectors are each different. In someembodiments, an AAV vector comprises an OTOF coding region, e.g., asdescribed herein. In some embodiments, a composition comprises an rAAVparticle comprising an AAV vector described herein. In some embodiments,the r AAV particle is encapsidated by an Anc80 capsid. In someembodiment, the Anc80 capsid comprises a polypeptide of SEQ ID NO: 109.

For example, in one embodiment, a therapeutic composition isadministered using a medical device capable of creating a plurality ofincisions in a round window membrane. In one embodiment, a medicaldevice includes a plurality of micro-needles. In one embodiment, amedical device includes a plurality of micro-needles including agenerally circular first aspect, wherein each micro-needle has adiameter of at least about 10 microns. In one embodiment, a medicaldevice includes a base and/or a reservoir capable of holding atherapeutic composition. In one embodiment, a medical device includes aplurality of hollow micro-needles individually including a lumen capableof transferring a therapeutic composition. In one embodiment, a medicaldevice includes a means for generating at least a partial vacuum.

As another example, a composition disclosed herein is administered usinga device and/or system specifically designed for intracochlear route ofadministration. In some embodiments, design elements of a devicedescribed herein may include: maintenance of sterility of injectedfluid; minimization of air bubbles introduced to the inner ear; abilityto precisely deliver small volumes at a controlled rate; deliverythrough the external auditory canal by the surgeon; minimization ofdamage to the round window membrane (RWM), or to inner ear, e.g.,cochlear structures beyond the RWM; and/or minimization of injectedfluid leaking back out through the RWM.

The devices, systems, and methods provided herein also describe thepotential for delivering a composition safely and efficiently into theinner ear, in order to treat conditions and disorders that would benefitfrom delivery of a composition disclosed herein to the inner ear,including, but not limited to, hearing disorders, e.g., as describedherein. As another example, by placing a vent in the stapes footplateand injecting through the RWM, a composition disclosed herein isdispersed throughout the cochlea with minimal dilution at the site ofaction. The development of the described devices allows the surgicaladministration procedure to be performed through the external auditorycanal in humans. The described devices can be removed from the earfollowing infusion of an amount of fluid into the perilymph of thecochlea. In subjects, the device may be advanced through the externalauditory canal, either under surgical microscopic control or along withan endoscope.

An exemplary device for use in any of the methods disclosed herein isdescribed in FIGS. 81-84 . FIG. 81 illustrates an exemplary device 10for delivering fluid to an inner ear. Device 10 includes a knurledhandle 12, and a distal handle adhesive 14 (for example, an epoxy suchas loctite 4014) that couples to a telescoping hypotube needle support24. The knurled handle 12 (or handle portion) may include kurlingfeatures and/or grooves to enhance the grip. The knurled handle 12 (orhandle portion) may be from about 5 mm to about 15 mm thick or fromabout 5 mm to about 12 mm thick, or from about 6 mm to about 10 mmthick, or from about 6 mm to about 9 mm thick, or from about 7 mm toabout 8 mm thick. The knurled handle 12 (or handle portion) may behollow such that fluid may pass through the device 10 during use. Thedevice 10 may also include a proximal handle adhesive 16 at a proximalend 18 of the knurled handle 12, a needle sub-assembly 26 (shown in FIG.82 ) with stopper 28 (shown in FIG. 82 ) at a distal end 20 of thedevice 10, and a strain relief feature 22. Strain relief feature 22 maybe composed of a Santoprene material, a Pebax material, a polyurethanematerial, a silicone material, a nylon material, and/or a thermoplasticelastomer. The telescoping hypotube needle support 24 surrounds andsupports a bent needle 38 (shown in FIG. 82 ) disposed therewithin.

Referring still to FIG. 81 , the stopper 28 may be composed of athermoplastic material or plastic polymer (such as a UV-cured polymer),as well as other suitable materials, and may be used to prevent the bentneedle 38 from being inserted too far into the ear canal (for example,to prevent insertion of bent needle 38 into the lateral wall or otherinner ear structure). Device 10 also may include a tapered portion 23disposed between the knurled handle 12 and the distal handle adhesive 14that is coupled to the telescoping hypotube needle support 24. Theknurled handle 12 (or handle portion) may include the tapered portion 23at the distal end of the handle portion 12. Device 10 may also includetubing 36 fluidly connected to the proximal end 16 the device 10 andacts as a fluid inlet line connecting the device to upstream components(for example, a pump, a syringe, and/or upstream components which, insome embodiments, may be coupled to a control system and/or power supply(not shown)). In some embodiments, the bent needle 38 (shown in FIG. 82) extends from the distal end 20, through the telescoping hypotubeneedle support 24, through the tapered portion 23, through the knurledhandle 12, and through the strain relief feature 22 and fluidly connectsdirectly to the tubing 36. In other embodiments, the bent needle 38fluidly connects with the hollow interior of the knurled handle (forexample, via the telescoping hypotube needle support 24) which in turnfluidly connects at a proximal end 16 with tubing 36. In embodimentswhere the bent needle 38 does not extend all the way through theinterior of the device 10, the contact area (for example, betweenoverlapping nested hypotubes 42), the tolerances, and/or sealantsbetween interfacing components must be sufficient to prevent therapeuticfluid from leaking out of the device 10 (which operates at a relativelylow pressure (for example, from about 1 Pascal to about 50 Pa, or fromabout 2 Pa to about 20 Pa, or from about 3 Pa to about 10 Pa)).

FIG. 82 illustrates a sideview of the bent needle sub-assembly 26,according to aspects of the present disclosed embodiments. Bent needlesub-assembly 26 includes a needle 38 that has a bent portion 32. Bentneedle sub-assembly 26 may also include a stopper 28 coupled to the bentportion 32. The bent portion 32 includes an angled tip 34 at the distalend 20 of the device 10 for piercing a membrane of the ear (for example,the RWM). The needle 38, bent portion 32, and angled top 34 are hollowsuch that fluid may flow therethrough. The angle 46 (as shown in FIG. 84) of the bent portion 32 may vary. A stopper 28 geometry may becyclidrical, disk-shaped, annulus-shaped, dome-shaped, and/or othersuitable shapes. Stopper 28 may be molded into place onto bent portion32. For example, stopper 28 may be positioned concentrically around thebent portion 32 using adhesives or compression fitting. Examples ofadhesives include an UV cure adhesive (such as Dymax 203A-CTH-F-T),elastomer adhesives, thermoset adhesives (such as epoxy orpolyurthethane), or emulsion adhesives (such as polyvinyl acetate).Stopper 28 fits concentrically around the bent portion 32 such thatangled tip 34 is inserted into the ear at a desired insertion depth. Thebent needle 38 may be formed from a straight needle using incrementalforming, as well as other suitable techniques.

FIG. 83 illustrates a perspective view of exemplary device 10 fordelivering fluid to an inner ear. Tubing 36 may be from about 1300 mm inlength (dimension 11 in FIG. 83 ) to about 1600 mm, or from about 1400mm to about 1500 mm, or from about 1430 mm to about 1450 mm. Strainrelease feature 22 may be from about 25 mm to about 30 mm in length(dimension 15 in FIG. 83 ), or from about 20 mm to about 35 mm inlength. Handle 12 may be about 155.4 mm in length (dimension 13 in FIG.83 ), or from about 150 mm to about 160 mm, or from about 140 mm toabout 170 mm. The telescoping hypotube needle support 24 may have two ormore nested hypotubes, for example three nested hypotubes 42A, 42B, and42C, or four nested hypotubes 42A, 42B, 42C, and 42D. The total lengthof hypotubes 42A, 42B, 42C and tip assembly 26 (dimension 17 in FIG. 83) may be from about 25 mm to about 45 mm, or from about 30 mm to about40 mm, or about 35 mm. In addition, telescoping hypotube needle support24 may have a length of about 36 mm, or from about 25 mm to about 45 mm,or form about 30 mm to about 40 mm. The three nested hypotubes 42A, 42B,and 42C each may have a length of 3.5 mm, 8.0 mm, and 19.8 mm,respectively, plus or minus about 20%. The inner-most nested hypotube(or most narrow portion) of the telescoping hypotube needle support 24may be concentrically disposed around needle 38.

FIG. 84 illustrates a perspective view of bent needle sub-assembly 26coupled to the distal end 20 of device 10, according to aspects of thepresent disclosed embodiments. As shown in FIG. 84 , bent needlesub-assembly 26 may include a needle 38 coupled to a bent portion 32. Inother embodiments, the bent needle 38 may be a single needle (forexample, a straight needle that is then bent such that it includes thedesired angle 46). Needle 38 may be a 33-gauge needle, or may include agauge from about 32 to about 34, or from about 31 to 35. At finergauges, care must be taken to ensure tubing 36 is not kinked or damaged.Needle 38 may be attached to handle 12 for safe and accurate placementof needle 38 into the inner ear. As shown in FIG. 84 , bent needlesub-assembly 26 may also include a stopper 28 disposed around bentportion 32. FIG. 84 also shows that bent portion 32 may include anangled tip 34 for piercing a membrane of the ear (for example, the RWM).Stopper 28 may have a height 48 of about 0.5 mm, or from about 0.4 mm toabout 0.6 mm, or from about 0.3 mm to about 0.7 mm. Bent portion 32 mayhave a length 52 of about 1.45 mm, or from about 1.35 mm to about 1.55mm, or from about 1.2 mm to about 1.7 mm. In other embodiments, the bentportion 32 may have a length greater than 2.0 mm such that the distancebetween the distal end of the stopper 28 and the distal end of theangled tip 34 is from about 0.5 mm to about 1.7 mm, or from about 0.6 mmto about 1.5 mm, or from about 0.7 mm to about 1.3 mm, or from about 0.8mm to about 1.2 mm. FIG. 84 shows that stopper 28 may have a geometrythat is cyclidrical, disk-shaped, and/or dome-shaped. A person ofordinary skill will appreciate that other geometries could be used.

The disclosure is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the disclosure should in no way beconstrued as being limited to the following examples, but rather shouldbe construed to encompass any and all variations that become evident asa result of the teaching provided herein.

Other assays, including those described in the Example section herein aswell as those that are known in the art, can also be used to evaluatethe auditory polypeptide nucleic acids and nucleic acid constructs ofthe disclosure.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentdisclosure and practice the claimed methods. The following workingexamples specifically point out various aspects of the presentdisclosure, and are not to be construed as limiting in any way theremainder of the disclosure.

EXAMPLES Example 1: Characterization of Human Otoferlin Gene, Homologs,Orthologs

The otoferlin gene and the corresponding mRNA are provided below.

Example 2: Construction of Viral Vectors

Recombinant AAV is generated by transfection with an adenovirus-freemethod as used by Xiao et al. J Virol 1999, 73(5):3994-4003. The cisplasmids with AAV ITRs, the trans plasmid with AAV Rep and Cap genes,and a helper plasmid with an essential region from an adenovirus genomeare co-transfected in 293 cells in a ratio of 1:1:2. The AAV vectorsused here express human otoferlin or mouse otoferlin under multiple dualvector strategies using the constructs described below. AAV serotypes 1,2, 3, 4, 5, 6, 7, 8, 9, rh8, rh10, rh39, rh43, and Anc80 are eachprepared to encapsulate three sets of otoferlin constructs to test (i) aconcatemerization-trans-splicing strategy, (ii) a hybridintronic-homologous recombination-trans-splicing strategy, and (iii) anexonic homologous recombination strategy, as summarized by Pryadkina etal. Meth Clin Devel 2015, 2:15009,

Example 3: Cloning OTOF Components

Oligo-dT and random-primed cDNA libraries are constructed from poly(A)+mRNA of human total fetus, adult brain, heart, kidney, and murine fetalheart as described by Yasunaga et al. Am J Genet 2000, 67:591-600.RACE-PCR experiments are performed on these libraries using linkerprimers and a series of primers selected from the otoferlin cDNAsequence. The PCR products are directly cloned into pGEM-T Easy vectorand sequenced. To isolate the human cDNA long form (7 kb), a reverseprimer (5′-TTCACCTGGGCCCGCA-GCATCCT-3′ (SEQ ID NO: 29)) is designed fromthe sequence encoding aa 63-70 of the initially reported short form ofotoferlin (Yasunaga et al., 1999) (GenBank 107403).

Total RNAs are extracted from mouse cochlea using the methods describedin Strenzke et al., EMBO J. 35(23):2519-2535, 2016. RT-PCR experimentsare performed in various murine and human RNA sources, according to theGeneAmp RNA PCR kit. Two primer pairs are used to reconstitute themurine cDNAs derived from the brain and the cochlea, one from the exon 15′-UTR (5′-AGGCGTGTGAGCCACACTCCACCA-3′ (SEQ ID NO: 30)) and exon 22(5′-CATAACCTCAGCTTGTCCCGAACA-3′ (SEQ ID NO: 31)), and the other from theexon 18-19 junction (5′-GGCCCCAGATCACGGACAGGAAC-3′ (SEQ ID NO: 32)) andexon 48 3′-UTR (5′-GGCCAGTACACCTGATTCACACT-3′ (SEQ ID NO: 33)). Toreconstitute the entire 5′ part of the human brain cDNA long form,primers derived from the 5′-UTR exon 1 (5′-GGAGGAGGCAGCGGCAGAGAAGA-3′(SEQ ID NO: 34)) and exon 22 (5′-TTCACCTGGGCCCGCAGCATCCT-3′ (SEQ ID NO:35)) are used.

For the concatemerization-trans-splicing strategy, two cassettes arecomposed such that the 5′ cassette includes a synthetic haircell-oriented promoter, a chimeric intron (β-globin), a consensus Kozaksequence, the exons 1 to 26 of otoferlin and the half intron 26 ofotoferlin (representing 3,836 bp, or the 3,494 bp of otoferlin cDNAcorresponding to exons 1 through 26, plus the first 342 bp of intron26), and the 3′ cassette includes the second half of intron 26 (342 bp),exons 27 to 48 (3,843 bp) of otoferlin, and a polyadenylation signalsequence. In some examples, the a hair cell-oriented promoter is notrequired for expression of an otoferlin protein in an auditory innerhair cell. For the intronic-homologous recombination-splicing strategy,the cassettes from the concatemerization-splicing strategy describedabove are modified such that the full length intron 26 of otoferlin isadded in the place of the half intron 26 in both plasmids.

For the exonic homologous recombination strategy, the two cassettes arecomposed such that the 5′ cassette includes a hair cell-orientedpromoter, a chimeric intron, a consensus Kozak sequence and the exons 1to 28 (the first 3,776 bp of the otoferlin cDNA), and the 3′ cassetteincludes the exons 23 to 48 (the final 4,446 bp of the otoferlin cDNA)and a polyadenylation signal sequence. The region of homology betweenthe two cassettes is 885 bp.

Example 4: Generating and Purifying Viral Particles

Recombinant AAV-1 is produced using a triple transfection protocol andpurified by two sequential cesium chloride (CsCl) density gradients, asdescribed by Pryadkina et al. Mol Ther 2015, 2:15009. At the end ofsecond centrifugation, 11 fractions of 500 μl are recovered from theCsCl Density Gradient tube and purified through dialysis in 1×PBS. Thefractions are analyzed by dot blot to determine those containing rAAVgenomes. The viral genome number (vg) of each preparation is determinedby quantitative real-time PCR-based titration method using primers andprobe corresponding to the ITR region of the AAV vector genome (Bartoliet al. Gene Ther 2006, 13:20-28).

Example 5: Formulation of Viral Particles

AAV produced at a titer of 1e14 vg/mL is prepared at dilutions of3.2e13, 1.0e13, 3.2e12, 1.0e12 vg/mL in artificial perilymph. Artificialperilymph is prepared by combining the following reagents, in mM: NaCl,120; KCl, 3.5; CaCl₂), 1.5; glucose, 5.5; HEPES, 20. The artificialperilymphis titrated with NaOH to adjust its pH to 7.5 (total Naconcentration of 130 mM) (Chen et al. J Controlled Rel 2005, 110:1-19).

Example 6: In Vitro Demonstration of OTOF mRNA and Protein Production(Anti-OTOF Antibody)

To confirm the AAV-OTOF vectors are capable of successfully transducingmammalian cells in vitro, human retinal epithelial cells and neonatemouse cochlear explants are incubated with AAV-OTOF at titers of 3.2e13,1.0e13, 3.2e12, 1.0e12 viral genome-containing particles (vg/mL) andassayed for levels of otoferlin DNA, mRNA and protein as describedpreviously (Duncker et al., 2013 J Neurosci 33(22):9508-9519. Antibodiesagainst mouse otoferlin are obtained from Abcam and used as described byEngel et al., 2006 Neurosci 143:837-849.

Example 7: Exemplary Device Description

The AAV-OTOF formulation is delivered to the cochlea using a specializedmicrocatheter designed for consistent and safe penetration of the RWM.The microcatheter is shaped such that the surgeon performing thedelivery procedure can enter the middle ear cavity via the externalauditory canal and contact the end of the microcatheter with the RWM.The distal end of the microcatheter is comprised of at least onemicroneedle with diameter of between 10 and 1,000 microns, which produceperforations in the RWM that are sufficient to allow AAV-OTOF to enterthe cochlear perilymph of the scala tympani at a rate of approximately 1μL/min, but heal without surgical repair. The remaining portion of themicrocatheter, proximal to the microneedle(s), is loaded with theAAV-OTOF/artificial perilymph formulation at a titer of approximately1e13 vg/mL. The proximal end of the microcatheter is connected to amicromanipulator that allows for precise, low volume infusions ofapproximately 1 TL/min.

Example 8: Animal Model 1: Aged Mice

Otoferlin rescue with cochlear delivery of AAV-OTOF is assessed in threeOTOF knockout mouse models (mouse models as described in Longo-Guess etal. Hearing Res 2007, 234(1-2):21-28; Roux et al. Cell 2006,127:277-289; and Reisinger et al., J. Neurosci. 31(13):4886-4895, 2011).Rescue experiments are tested in neonate (P1), juvenile (P6 or P12) andadult (P42) mice, in order to evaluate the postnatal treatment windowrelative to stage of cochlear development.

Baseline auditory brainstem response (ABR) and distortion productoptoacoustic emissions (DPOAEs) are measured in the juvenile and adultmice (n=32), bilaterally, to assess pre-treatment inner hair cell (IHC)and outer hair cell (OHC) function. All animals are expected to displaythe characteristic audiometric profile of otoferlin dysfunction—i.e.,abnormal ABRs across tested sound frequencies but normal DPOAEs,indicative of dysfunctional IHC signal transduction and normal OHCfunction (Yasunaga et al. 2000, Am J Hum Genet 67:591-600).

Following baseline ABR and DPOAE measurements, 0.3 uL of AAV1-OTOF attiters of 1.0e13, 3.2e12, and 1.0e12 vg/mL is injected into the leftscala tympani of the juvenile and adult mice (n=32), as described below.Equivalent titers are injected in the left scala tympani of the neonatalmice, but at a volume of 0.2 uL (n=16) (surgical procedure describedbelow). Each animals' right ear is left as an untreated control. ABR andDPOAE measurements are taken again bilaterally in the juvenile and adultanimals 1, 5 and 10 days following the surgical procedure. At 4 weeks(n=24) and 12 weeks (n=24) post-procedure, additional bilateral ABR andDPOAE measurements are taken from all animals in the neonate, juvenileand adult groups (total n=48), and the animals are subsequentlysacrificed and their cochleae removed.

In half of the sacrificed animals (n=4 from each of the 4 week and 12week post-treatment groups), immunostaining is performed to identifyhair cell structures and to assess OTOF protein expression along thecochlear sensory epithelium. Antibodies against markers for hair cells(Myo7a), supporting cells (Sox2) and otoferlin are used as describedpreviously (Duncker et al. 2013, J Neurosci 33(22):9508-9519. At thebasal, middle and apical turns of the organ of corti, total numbers ofhair cells and those hair cells expressing OTOF were counted within 200um regions; the entire length of the organ of corti was divided intothree pieces of equal length, designated the basal, middle and apicalregions.

In the remaining half of the sacrificed animals (remaining 4 animalsfrom each of the 4 week and 12 week post-treatment groups), cochleartissue samples are collected from the same basal, middle and apicalregions as described above, and assayed for otoferlin mRNA transcript asdescribed previously (Duncker et al. 2013, J Neurosci 33(22):9508-9519,Heidrych et al. 2008, Hum Mol Genet 17:3814-3821, Heidrych et al., 2009,Hum Mol Genet 18:2779-2790).

Example 9: Animal Model 1A: Surgical Method in Aged Mice

AAV-OTOF prepared in artificial perilymph is administered to the scalatympani in mice as described by Shu et al. 2016 (Shu Yilai, Tao Yong,Wang Zhengmin, Tang Yong, Li Huawei, Dai Pu, Gao Guanping, and ChenZheng-Yi. Human Gene Therapy. June 2016, ahead of print.doi:10.1089/hum.2016.053). Six-week-old male mice are anesthetized usingan intraperitoneal injection of xylazine (20 mg/kg) and ketamine (100mg/kg). Body temperature is maintained at 37° C. using an electricheating pad. An incision is made from the right post-auricular regionand the tympanic bulla is exposed. The bulla is perforated with asurgical needle and the small hole is expanded to provide access to thecochlea. The bone of the cochlear lateral wall of the scala tympani isthinned with a dental drill so that the membranous lateral wall is leftintact. A Nanoliter Microinjection System in conjunction with glassmicropipette is used to deliver a total of approximately 300 nL ofAAV-OTOF in artificial perilymph to the scala tympani at a rate of 2nL/second. The glass micropipette is left in place for 5 minutespost-injection. Following cochleostomy and injection, the opening in thetympanic bulla is sealed with dental cement, and the muscle and skin aresutured. The mice are allowed to awaken from anesthesia and their painis controlled with 0.15 mg/kg buprenorphine hydrochloride for 3 days.

Example 10: Animal Model 2: Reciprocating Micropump in Guinea Pig

Surgical Procedure

AAV-OTOF prepared in artificial perilymph is administered to guinea pigsto assess distribution and toxicity following intracochlear deliverywith a reciprocating micropump as described by Tandon et al. Lab Chip2015 (DOI: 10.1039/c51c01396h). Male guinea pigs weighing approximately350 g each (n=16) are anesthetized with a combination of pentobarbitalsodium (Nembutal; 25 mg kg-1, injected intraperitoneally), fentanyl (0.2mg kg-1, intramuscularly), and haloperidol (10 mg kg-1,intramuscularly). Lidocaine with epinephrine is given subcutaneously atthe incision site as a topical anesthetic. Using a dorsal approach, a 5mm diameter hole is made in the bulla and a cochleostomy is createdapproximately 0.5 mm distal to the round window membrane. The cannula ofthe micropump (described below) is inserted into the cochleostomy,threaded into the cochlea 3 mm apically, and glued to the bulla with acommon cyanoacrylate glue. For compound action potential (CAP)measurements, a perfluoroalkoxy-alkane-insulated silver wire electrode(203 μm uncoated diameter) is inserted near the round window niche andglued to the bulla.

Procedures for measurement of distortion product otoacoustic emissions(DPOAEs) and CAPs are performed as previously described in Tandon et al.Biomed Microdevices 2015, 17:3-21. DPOAEs are measured before and afterthe cochleostomy procedure at the characteristic frequencies: 32, 24,16, 12, 8, 5.6, 4, and 2.78 kHz in order to monitor any damage thatoccurs as a result of the surgery.

Micropump Description

AAV-OTOF at a maximum titer of 1e14 vg/mL is administered to the guineapig using a micropump as described by Tandon et al. Lab Chip 2015 (DOI:10.1039/c51c01396h). The micropump system has 4 selectable ports. Theseports are connected to: (i) a large fluidic capacitor used forartificial perilymph storage; (ii) an outlet that connects to thecochlea; (iii) the outlet from an integrated AAV-OTOF reservoir; (iv)the inlet to the integrated AAV-OTOF reservoir. Each port is fluidicallyconnected to a central pump chamber, and each is individually addressedwith a valve. The sequence of events for reciprocating AAV-OTOF deliveryis as follows: (i) an internal AAV-OTOF-refresh loop is run,transferring AAV-OTOF from the AAV-OTOF reservoir into the maininfuse-withdraw line; (ii) AAV-OTOF is infused into the cochlea and someartificial perilymph is drained from the artificial perilymph storagecapacitor; (iii) the first two steps can be repeated several times foradditional doses; (iv) after the AAV-OTOF has been allowed to diffusefor some time, a volume of perilymph is withdrawn from the cochlea thatis equal to the volume infused in steps (i)-(iii), refilling theartificial perilymph storage capacitor. This process results in netdelivery of drug with zero net fluid volume added to the cochlea.

The fluidic capacitors in the micropump are cylindrical chambers whoseceilings are a thin (25.4 m), flexible, polyimide membrane. The pumpchamber has a diameter of 3.5 mm, the fluidic storage capacitor has adiameter of 14 mm, and all of the remaining capacitors have diameters of4 mm. The same membrane is deflected to block flow at each of thevalves. The valve chambers have diameters of 3.1 mm. The serpentinechannel that comprises the drug reservoir has a square cross section ofwidth 762 m and a length of 410 mm for a total volume of 238 μL. All ofthe other microchannels in the pump have a width of 400 μm and a heightof 254 μm.

Acute Drug Delivery in Guinea Pigs

The micropump is loaded with AAV-OTOF and artificial perilymph, and thecannula inserted into a cochleostomy made in the region of the cochleabetween the locations with characteristic frequency sensitivity of 24and 32 kHz, and threaded apically 3 mm, terminating in the 12-16 kHzregion. Baseline DPOAE and CAP hearing tests are performed prior to thestart of AAV-OTOF/artificial perilymph infusion. The pump is thenactivated and approximately 1 μL of artificial perilymph is infusedevery 5 min until a total of approximately 10 μL of artificial perilymphis delivered to the cochlea. After a 20 min wait time, approximately 10μL of perilymph is withdrawn from the cochlea. AAV-OTOF delivery is theninitiated at a rate of approximately 1 μL every 5 min until a total ofapproximately 10 μL of fluid is delivered.

Animals are sacrificed at 1 week, 1 month, 3 months, and 6 monthspost-treatment (n=4 per group) and their cochleae extracted. Extent ofAAV transduction and OTOF expression along the organ of Corti isassessed via immunostaining with anti-OTOF antibodies. Antibodiesagainst markers for hair cells (Myo7a) and supporting cells (Sox2) areused to quantify IHCs, OHCs, supporting cells and stereociliamorphology. Annexin V staining is used to assess evidence of apoptosisin cells along the cochlear sensory epithelium.

Example 11: Animal Model 3: Large Animal Tox in Sheep

AAV-OTOF prepared in artificial perilymph is administered to juvenilesheep to assess distribution and toxicity following delivery to thecochlea via trans-RWM infusion. Baseline auditory brainstem response(ABR) and distortion product optoacoustic emissions (DPOAEs) aremeasured in female sheep at 3 months of age (n=40), bilaterally, toassess pre-treatment inner hair cell (IHC) and outer hair cell (OHC)function. Following baseline ABR and DPOAE measurements, 20 uL ofAAV1-OTOF at titers of 1.0e14, 3.2e13, 1.0e13 and 3.2e12 vg/mL isinjected into the left scala tympani of the sheep (n=10 per group). Eachanimal's right ear is left as an untreated control. ABR and DPOAEmeasurements are taken again bilaterally 1, 5 and 10 days following thesurgical procedure. At 6 months post-procedure, additional bilateral ABRand DPOAE measurements are taken from all animals, and the animals aresubsequently sacrificed and their cochleae removed.

In half of the sacrificed animals (n=5 from each of the dose cohorts),immunostaining is performed to identify hair cell structures and toassess OTOF protein expression along the cochlear sensory epithelium.Antibodies against markers for hair cells (Myo7a), supporting cells(Sox2) and otoferlin are used as described previously (Duncker et al.2013, J Neurosci 33(22):9508-9519). At the basal, middle and apicalturns of the organ of corti, total numbers of hair cells and those haircells expressing OTOF are counted within 200 um regions.

In the remaining half of the sacrificed animals (remaining 5 animalsfrom each dose cohort), cochlear tissue samples are collected from thesame basal, middle and apical regions as described above, and assayedfor otoferlin mRNA transcript as described previously (Duncker et al.2013, J Neurosci 33(22):9508-9519, Heidrych et al. 2008, Hum Mol Genet17:3814-3821, Heidrych et al., 2009, Hum Mol Genet 18:2779-2790).

Example 12: Animal Model 3A: CRISPR Generated Transgenic Large AnimalModel (Sheep)

Generation of Plasmid Co-Expressing Cas9 and sgRNA

The pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene plasmid #42230) isdigested with BsbI, dephosphorylated using Antartic Phosphatase, and thelinearized vector is gel purified. To generate the bicistronic vector(pX330-cas9-OTOF) expressing Cas9 and sgRNA against OTOF, a pair ofoligos for targeting otoferlin exon 1 is annealed, phosphorylated andligated to a linearized vector (Cong et al. 2013 Science339(6121):819-23).

Genome Editing Assay in Cells

The A15 astroglial sheep cell line (Vilette et al., 2000 In Vitro CellDev Biol Anim 36(1):45-9) is maintained in DMEM in 10% Fetal BovineSerum, 2 mM glutamine, 1% sodium pyruvate and 1%penicillin/streptomycin. Cells are transfected in 24-well plates with 2μg of pX330-cas9-OTOF co-expressing Cas9 and sgRNA against otoferlinusing lipofectamine LTX reagent. Three days later, genomic DNA fromtransfected cells is extracted and quantified using a NanoDrop2000spectrophotometer, measuring

A2601A280 and A2601A230 Ratios to Account for Sample Purity

Gene mutation activity of sgRNA sequence at the target locus of OTOFexon 1 is quantified using the T7EI mismatch detection assay. DNAsequence of interest is PCR-amplified with a high-fidelity polymerase(Herculase II fusion polymerase) using specific primers. The resultantPCR product is then denatured and slowly re-annealed (95° C., 2 min; 95°C. to 85° C., −2° C./sec; 85° C. to 25° C., −1° C./sec) to producehomoduplex/heteroduplex mix. This is then digested by 5 U of T7EIrestriction enzyme at 37° C. for 30 minutes. Digestion products areseparated by 2% agarose gel electrophoresis. The ratio of cleaved touncleaved products is used to calculate NHEJ frequency as previouslydescribed using Image J software (Menoret et al. 2011 Advanced protocolsfor Animal Transgenesis. An ISTT Manual. Heidelberg: Springer. p117-36).NHEJ frequency is calculated as % gene modification=100×(1−(1−fractioncleaved){circumflex over ( )}(½).

Production of sgRNA and Cas9 mRNA

As described previously (Bellec et al. 2015, Current Gene Ther), T7promoter is added to sgRNA template by PCR amplification ofpX330-cas9-OTOF plasmid. The PCR product is purified using NucleoSpinGel and PCR Clean-up. It is used as the template for in vitrotranscription using MEGAshortscript T7 kit according to themanufacturer's manual. Following completion of transcription, DNase Itreatment is performed.

The Cas9 mRNA is transcribed using PmeI-digested Cas9 expression JDS246plasmid (Addgene plasmid #43861) and the mMESSAGE mMACHINE T7 ULTRATranscription Kit according to the manufacturer's manual. Followingcompletion of transcription, the poly(A) tailing reaction and DNase Itreatment are performed. Both the Cas9 mRNA and the sgRNAs are purifiedusing MEGAclear kit and eluted in elution buffer.

In Vitro Production of Embryos

The embryos are produced by in vitro fertilization according to routineprocedure as described previously (Crispo et al. 2014 Transgenic Res,24(1):31-41). Briefly, ovaries from slaughterhouse are transported tothe laboratory and cumulus oocyte complexes (COCs) are aspirated inrecovery medium. The selected COCs are placed in maturation medium for24 h in 5% C02 in humidified air atmosphere at 39° C. Then, expandedCOCs are inseminated in 100 l drops with 1×106 dose of frozen-thawedsemen selected by ascendant migration on a swim up method. Fertilizationis carried out in 5% CO2 with humidified atmosphere at 39° C. for 22 h.

Microinjection into Zygotes

Soon after fertilization, 572 presumptive zygotes are randomly assignedto three experimental groups to be microinjected (CRISPR group, n=200;and Buffer group, n=200) or not (Control group, n=200). Microinjectionof CRISPR group is performed into the cytoplasm with 5 ng/μl of sgRNAand 20 ng/μl of Cas9 mRNA diluted in injection buffer (10 mM Tris pH7.5, 0.1 mM EDTA), while Buffer group is injected with the sameprocedure but with buffer alone. Lastly, injected and non-injectedembryos are transferred to culture medium under mineral oil, in 5% CO2,5% 02 and 90% N2 in humidified atmosphere at 39° C. Cleavage rate on Day2 (cleaved zygotes per total oocytes) and development rate on Day 6(morulae and blastocysts per total oocytes) are recorded for allexperimental groups. After Day 6, DNA from 20 CRISPR group embryos areanalyzed by Sanger sequencing to detect the mutation at the OTOF genelevel.

To determine the in vivo efficiency of the system, 53 blastocystsproduced by CRISPR/Cas9 zygote microinjection are transferred to 29recipient females. Only early blastocysts, blastocysts and expandedblastocysts classified as excellent or good (i.e. Grade 1 as defined inStringfellow et al. 2010, Manual of the International Embryo TransferSociety) are transferred on Day 6 after fertilization. Embryo transferis performed by minimally invasive surgery assisted by laparoscopy toplace the embryos into the cranial side of the ipsilateral uterine hornto the corpus luteum. Recipient ewes are previously synchronized to beon Day 6 of the estrous cycle using a standard protocol to controlovulation described previously, as described by Menchaca et al. 2004,Reprod Fertil Dev. 16(4):403-413.

Monitoring of Fetuses and Lambs

Pregnancy diagnosis and fetal development are performed on Day 30 and105, respectively, by using B-mode ultrasonography equipped with a 5 and3.5 MHz probe. Day 0 of the experiment is defined as the moment ofembryo fertilization. Several parameters are measured to study thedevelopment of fetuses at Day 105 of gestation: thoracic diameter,biparietal diameter, occipitonasal length and heart rate. At delivery,length of gestation, gender, rectal temperature, heart and respiratoryrates, body weight, thoracic perimeter, biparietal diameter, crown-rumpand occipitonasal length, height at withers, height at hips, width athips and width at chest were recorded. Body weight and morphometricvariables are determined at birth, and 15, 30 and 60 days later.

Identification and Genotyping of Transgenic Animals

Samples from skin and limb muscle of the lambs are taken seven daysafter birth and T7EI assay, western blot test and histology examinationsare performed in order to identify and characterize KO founders andoff-target sites. Total DNA is isolated from skin biopsies for allanimals and from muscle for some animals. Samples are analyzed usingcapillary electrophoresis. Genotyping of OTOF exon 1 is performed bydirect sequencing of PCR amplicons and in muscle biopsies by additionalsequencing of isolated bacterial clones with individual ampliconsequences.

Analysis of Otoferlin Expression

Western blotting is performed to determine the presence of myostatin inthe muscle fiber. Equal amounts of total proteins are run on 12% (v/v)gel electrophoresis and electrophoretically transferred to a PVDFmembrane. Monoclonal mouse anti-otoferlin antibody is used in thewestern blotting. The washed membranes are incubated with 1:50000dilution of secondary antibody linked to horseradish peroxidase (HPR).HPR activity is detected using western blot chemiluminescence.

AAV-OTOF Rescue Therapy in Transgenic Sheep Model

AAV-OTOF prepared in artificial perilymph is administered to OTOFknockout transgenic sheep to assess the ability to restore normalhearing function following delivery to the cochlea via trans-RWMinfusion. Baseline auditory brainstem response (ABR) and distortionproduct optoacoustic emissions (DPOAEs) are measured in female sheep at3 months of age (n=30), bilaterally, to assess pre-treatment inner haircell (IHC) and outer hair cell (OHC) function. Following baseline ABRand DPOAE measurements, 20 uL of AAV1-OTOF at titers of 1.0e14, 3.2e13and 1.0e13 vg/mL is injected into the left scala tympani of the sheep(n=10 per group). Each animal's right ear is left as an untreatedcontrol. ABR and DPOAE measurements are taken again bilaterally 1, 5 and10 days following the surgical procedure. At 6 months post-procedure,additional bilateral ABR and DPOAE measurements are taken from allanimals, and the animals are subsequently sacrificed and their cochleaeremoved.

In half of the sacrificed animals (n=5 from each of the dose cohorts),immunostaining is performed to identify hair cell structures and toassess OTOF protein expression along the cochlear sensory epithelium.Antibodies against markers for hair cells (Myo7a), supporting cells(Sox2) and otoferlin are used as described previously (Duncker et al.2013, J Neurosci 33(22):9508-9519). At the basal, middle and apicalturns of the organ of corti, total numbers of hair cells and those haircells expressing OTOF are counted within 200 um regions.

In the remaining half of the sacrificed animals (remaining 5 animalsfrom each dose cohort), cochlear tissue samples are collected from thesame basal, middle and apical regions as described above, and assayedfor otoferlin mRNA transcript as described previously (Duncker et al.2013, J Neurosci 33(22):9508-9519, Heidrych et al. 2008, Hum Mol Genet17:3814-3821, Heidrych et al., 2009, Hum Mol Genet 18:2779-2790).

Example 13: Human Clinical Example (Pediatric Treatment)

The subject is put under general anesthesia. The surgeon approaches thetympanic membrane from external auditory canal, makes a small incisionat the inferior edge of the external auditory canal where it meets thetympani membrane, and lifts the tympanic membrane as a flap to exposethe middle ear space. A surgical laser is used to make a small opening(approximately 2 mm) in the stapes footplate. The surgeon thenpenetrates the round window membrane with a microcatheter loaded with asolution of AAV-OTOF prepared in artificial perilymph at a titer of 1e13vg/mL. The microcatheter is connected to a micromanipulator that infusesapproximately 20 uL of the AAV-OTOF solution at a rate of approximately1 uL/min. At the conclusion of the AAV-OTOF infusion, the surgeonwithdraws the microcatheter and patches the holes in the stapes footplate and RWM with a gel foam patch. The procedure concludes withreplacement of the tympanic membrane flap.

Example 14: Non-Invasive Prenatal Testing of Maternal Blood to DetectOTOF Mutation

Maternal blood samples (20-40 mL) are collected into Cell-free DNAtubes. At least 7 mL of plasma is isolated from each sample via a doublecentrifugation protocol of 2,000 g for 20 minutes, followed by 3,220 gfor 30 minutes, with supernatant transfer following the first spin.cfDNA is isolated from 7-20 mL plasma using a QIAGEN QIAmp CirculatingNuclei Acid kit and eluted in 45 μL TE buffer. Pure maternal genomic DNAis isolated from the buffy coat obtained following the firstcentrifugation.

By combining thermodynamic modeling of the assays to select probes withminimized likelihood of probe-probe interaction with amplificationapproaches described previously (Stiller et al. 2009 Genome Res19(10):1843-1848), multiplexing of 11,000 assays can be achieved.Maternal cfDNA and maternal genomic DNA samples are pre-amplified for 15cycles using 11,000 target-specific assays and an aliquot is transferredto a second PCR reaction of 15 cycles using nested primers. Samples areprepared for sequencing by adding barcoded tags in a third 12-cycleround of PCR. The targets include SNPs corresponding to the 100mutations in chromosome 2 known to lead to otoferlin loss-of-function(Zhang et al. 2016 Clin Genetics January 27). The amplicons are thensequenced using an Illumina HiSeq sequencer. Genome sequence alignmentis performed using commercially available software.

Example 15: Alternative Examples (mRNA, Single Viral Vector, Non-ViralVectors)

Single Viral Vector Preparation

Mouse otoferlin lacking the N-terminal domains can rescue the knockdownphenotype in otoferlin knockout zebrafish (Chatterjee et al. Mol CellBiol 2015, 35(6):1043-1054). However, in mammals, missense mutations inthe C2B and C2C domains have been linked to hearing loss (Longo-Guess etal., 2007 Hear Res, 234:21-28; Mirghomizadeh et al., 2002 Neurobiol Dis10:157-164), suggesting that these domains are essential to the normalhearing-related function of otoferlin in higher species. The C2A domainof mammalian otoferlin does not bind Ca²⁺, whereas all other C2 domainsbind Ca²⁺ with moderate (20-50 μM) or low (400-700 μM) affinity in theabsence of acidic lipids. The C2D and C2E domains bind Ca²⁺ as well asphosphatidylserine (PS) in a Ca²⁺-dependent manner. A cDNA is producedthat encodes a truncated form of otoferlin lacking the C2A, C2D and C2Edomains. This cDNA is suitable for packaging in an AAV vector.

The truncated otoferlin construct (OTOFΔC2ADE) is derived and clonedfrom an original wildtype otoferlin plasmid encoding the full OTOF gene,as described by Padmanarayana et al. 2014 Biochem 53:5023-5033. Deletionof the coding region of the C2 domains is performed by PCR mutagenesisusing domain-spanning oligonucleotides and a QuikChange site-directedmutagenesis kit applying the double mutagenic primer approach. Briefly,the PCR is performed as follows: 95° C. for 3 minutes; 18 cycles at 95°C. for 15 seconds, 65° C. for 1 minute, and 68° C. for 12 minutes; and68° C. for 7 minutes. The PCR product is digested with DpnI, cloned intothe DSC-B vector, and transformed into DH5alpha or XL10-Gold bacterialcells. Plasmid DNA is isolated by mini preparations and subsequentlysequenced.

A plasmid containing a CBA promoter, a chimeric intron (β-globin), aconsensus Kozak sequence, the OTOFΔC2ADE cDNA and a polyadenylationsignal sequence is used for the AAV construct. Recombinant AAV isgenerated by transfection with an adenovirus-free method as used by Xiaoet al. J Virol 1999, 73(5):3994-4003. The cis plasmids with AAV ITRs,the trans plasmid with AAV Rep and Cap genes, and a helper plasmid withan essential region from an adenovirus genome are co-transfected in 293cells in a ratio of 1:1:2. AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, rh8,rh10, rh39, rh43, and Anc80 are each prepared to encapsulate theOTOFΔC2ADE cDNA construct.

CELiD Preparation

The otoferlin gene is prepare for non-viral gene transfer as describedby Li et al. 2013, PLoS ONE 8(8):e69879. First, Spodoptera frugiperdaSf9 cells are grown in suspension in serum-free media. The blasticidin-Sdeaminase (bs) gene is PCR-amplified from pIB/V5-His/CAT using thefollowing primer pair:

(SEQ ID NO: 36) 5′-ATAAGCTTACGCTCAGTGGAACGAAAAC-3′ and (SEQ ID NO: 37)5′-ATAAGCTTGACGTGTCAGTGTCAGTCCTGCTCCT-3′.The 865 bp PCR product is digested with HindIII and ligated intoHindIII-digested pFBGR. Sf9 cells are transfected with pFBGR-bsd usingCellfectin Transfection Reagent. At three days post-transfection,antibiotic-resistant cells are selected by the addition of blasticidin-SHCl (50 μg/mL) to the growth medium. After two weeks in selectivemedium, blasticidin-resistant (bsd^(r)) clones are derived bysingle-cell dilution or direct colony transfer techniques. The bsd^(r)clones are expanded in insect cell culture medium supplemented with 10%FBS and blasticidin-S HCl (10 μg/mL) for 2 to 3 additional passages,then returned to serum-free medium with 10 μg/mL blasticidin-S HCl.After an additional 12 passages, blasticidin-S HCl is omitted from themedium and the cell lines are expanded for analysis. For functionalscreening, clonal Sf9/ITR-OTOF cell lines are infection (MOI=5) with arecombinant baculovirus, Bac-Rep, expressing the AAV type 2 Rep78 andRep52 proteins and analyzed for induced OTOF expression. ClonalSf9/ITR-OTOF cells with the highest levels of OTOF expression areexpanded for CELiD-OTOF DNA preparation.

Clonal Sf9/ITR-OTOF cells are seeded at 2e6 cells/mL and infected withBac-Rep (MOI=1 to 3). Cell viability and diameter are monitored dailyuntil the cell diameter increased to 18-20 μm (uninfected cell diameter14-15 μm), indicating that the cells are in the late stages of the viralinfection. Extrachromosomal DNA is extracted from the Bac-Rep-infected,Sf9/ITR-GFP cells using a commercially available plasmid isolation kit.CELiD production is monitored by agarose gel electrophoresis andethidium bromide staining of extrachromosomal DNA. CELiD DNA is producedin parental Sf9 cells by co-infection with two separate baculovirusexpression vectors (BEV): Bac-Rep and a second BEV bearing anITR-flanked transgene, such as Bac-OTOF. Infected Sf9 cells areharvested once the mean cell diameter increases by 4-5 μm and thepercent viability decreases to 80-90%. CELiD DNA is isolated using acommercially available plasmid purification kit.

Clonal Sf9/ITR-OTOF cells are inoculated with various amounts of Bac-Repstock. Cells are periodically harvested and extrachromosomal DNA isrecovered using a commercially available DNA isolation kit. ExtractedDNA is examined by either agarose gel electrophoresis or by PCR withOTOF-specific primer pairs for quantitative determination of CELiD DNAamounts. For western blotting, cell proteins are fractionated bySDS-polyacrylamide gel electrophoresis and transferred to nitrocellulosemembranes. The membranes are incubated in blocking buffer (BB) composedof 5% non-fat dry milk (w:v) in phosphate-buffered saline plus 0.05%Tween-20 (PBST) for 1 hr at ambient temperature with orbital agitation.After washing the membranes in wash buffer (WB) composed of 3% non-fatdry milk in PBST, membranes are incubated with the appropriate primaryantibody solution (diluted in BB) either at ambient temperature (1 hr)or 4° C. (overnight) with continuous orbital agitation. The followingprimary antibodies and dilution ratios are used: anti-AAV Rep mousemonoclonal antibody (mAb); 2. anti-baculovirus envelope glycoproteingp64 mouse mAb; anti-OTOF mouse mAb. After incubation, primary antibodysolutions are removed and membranes are washed in WB (3×5 mins).Non-conjugated mAbs are incubated with secondary antibody solution(goat, anti-mouse horseradish peroxidase (RP)-conjugate for 1 hr, andthen washed with WB as above. HRP activity is detected by enhancedchemiluminescence (ECL).

Lipid Nanoparticles

Otoferlin cDNA or mRNA is encapsulated in poly(lactic-co-glycolic acid)nanoparticles by the double-emulsion solvent evaporation methoddescribed previously (O'Donnell and McGinity 1997 Adv Drug Delivery Rev28(1):25-42), and in lipid nanoparticles (Pezzoli et al. 2013 MethodsMolBiol 1025:269-279). Briefly, solid lipid nanoparticles can begenerated from a microemulsion using Precirol ATO-5 and stearylamine asthe cationic lipid. 500 mg of Precirol ATO-5 is heated to 10° C., aboveits melting point, and 10 mL of a hot aqueous solution of poloxamer andstearylamine in different proportions (1/1.25; 1/1.87; 1/3.12; 1/4.37and 1/5) is added. The sample is stirred for 30 minutes at 14,000 rpm.The nanoparticles are generated by dispersing the hot microemulsion incold water (2-5° C.) in an emulsion:water ratio of 1:5. To recovernanoparticles, the resultant suspension is centrifuged for three timesat 3,000 rpm for 20 minutes at a temperature of 20° C., reconstitutingthe precipitate after centrifugation. Cationic solid lipid nanoparticlesare lyophilized by being added an aqueous solution of cryoprotectant (5%mannitol) in a 1:2 (SLN:mannitol) ratio. The freezing temperature is setat −40° C. in the lyophilizer and samples are kept at this temperaturefor 2 hours. Lyophilization temperature is then set to 25° C. at apressure of 0.2-0.4 mBa for 48 hours. A solution of the OTOF cDNAplasmid is prepared to a concentration of 2 μg/μL. A 25 μL aliquot ofthe plasmid DNA solution is then added to different volumes of thecationic SLN suspension to obtain ratios of between 15:1 and 1:1(SLN:OTOF) by stirring.

Modified RNA

Polynucleotides, primary constructs mRNA (or modified mRNA, or “mmRNA”)for use in accordance with the disclosure may be prepared according toany available technique including, but not limited to chemicalsynthesis, enzymatic synthesis, which is generally termed in vitrotranscription (IVT), or enzymatic or chemical cleavage of a longerprecursor, etc. Methods of synthesizing RNAs are known in the art (see,e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: TRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

The process of design and synthesis of the primary constructs of thedisclosure generally includes the steps of gene construction, mRNAproduction (either with or without modifications) and purification. Inthe enzymatic synthesis method, a target polynucleotide sequenceencoding the polypeptide of interest is first selected for incorporationinto a vector, which will be amplified to produce a cDNA template.Optionally, the target polynucleotide sequence and/or any flankingsequences may be codon optimized. The cDNA template is then used toproduce mRNA through in vitro transcription (IVT). After production, themRNA may undergo purification and clean-up processes. The steps of whichare provided in more detail below.

Gene Construction

The step of gene construction may include, but is not limited to genesynthesis, vector amplification, plasmid purification, plasmidlinearization and clean-up, and cDNA template synthesis and clean-up.

Gene Synthesis

Once a polypeptide of interest, or target, is selected for production, aprimary construct is designed. Within the primary construct, a firstregion of linked nucleosides encoding the polypeptide of interest may beconstructed using an open reading frame (ORF) of a selected nucleic acid(DNA or RNA) transcript. The ORF may comprise the wild type ORF, anisoform, variant or a fragment thereof. As used herein, an “open readingframe” or “ORF” is meant to refer to a nucleic acid sequence (DNA orRNA) that encodes a polypeptide of interest. ORFs often begin with thestart codon, ATG, and end with a nonsense or termination codon orsignal. Further, the nucleotide sequence of the first region may becodon optimized. Codon optimization methods are known in the art and maybe useful in efforts to achieve one or more of several goals. Thesegoals include to match codon frequencies in target and host organisms toensure proper folding, bias GC content to increase mRNA stability orreduce secondary structures, minimize tandem repeat codons or base runsthat may impair gene construction or expression, customizetranscriptional and translational control regions, insert or removeprotein trafficking sequences, remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites), add, remove orshuffle protein domains, insert or delete restriction sites, modifyribosome binding sites and mRNA degradation sites, adjust translationalrates to allow the various domains of the protein to fold properly, orreduce or eliminate problematic secondary structures within the mRNA.Codon optimization tools, algorithms and services are known in the art.Non-limiting examples include services from GeneArt (Life Technologies)and DNA2.0 (Menlo Park Calif.) and/or proprietary methods. In oneembodiment, the ORF sequence is optimized using optimization algorithms.Codon options for each amino acid are known in the art.

Stop Codons

In one embodiment, the primary constructs of the present disclosure mayinclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon may be selected from TGA, TAA and TAG. In oneembodiment, the primary constructs of the present disclosure include thestop codon TGA and one additional stop codon. In a further embodiment,the additional stop codon may be TAA. In another embodiment, the primaryconstructs of the present disclosure include three stop codons.

Vector Amplification

The vector containing the primary construct is then amplified and theplasmid isolated and purified using methods known in the art such as,but not limited to, a maxi prep using the Invitrogen PURELINK™ HiPureMaxiprep Kit (Carlsbad, Calif.).

Plasmid Linearization

The plasmid may then be linearized using methods known in the art suchas, but not limited to, the use of restriction enzymes and buffers. Thelinearization reaction may be purified using methods including, forexample Invitrogen's PURELINK® PCR Micro Kit (Carlsbad, Calif.), andHPLC based purification methods such as, but not limited to, stronganion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). The purification methodmay be modified depending on the size of the linearization reactionconducted. The linearized plasmid is then used to generate cDNA for invitro transcription (IVT) reactions.

cDNA Template Synthesis

A cDNA template may be synthesized by having a linearized plasmidundergo polymerase chain reaction (PCR). Primer-probe design for anyamplification is within the skill of those in the art. Probes may alsocontain chemically modified bases to increase base-pairing fidelity tothe target molecule and base-pairing strength. Such modifications mayinclude 5-methyl-Cytidine, 2,6-di-amino-purine, 2′-fluoro,phosphoro-thioate, or locked nucleic acids.

mRNA Production

The process of mRNA or mmRNA production may include, but is not limitedto, in vitro transcription, cDNA template removal and RNA clean-up, andmRNA capping and/or tailing reactions.

In Vitro Transcription

The cDNA produced above may be transcribed using an in vitrotranscription (IVT) system. The system typically comprises atranscription buffer, nucleotide triphosphates (NTPs), an RNaseinhibitor and a polymerase. The NTPs may be manufactured in house, maybe selected from a supplier, or may be synthesized as described herein.The NTPs may be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasemay be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate modified nucleic acids.

5′ Capping

The 5′ cap structure of an mRNA is involved in nuclear export,increasing mRNA stability. It binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns during mRNA splicing. Endogenous mRNAmolecules may be 5′-end capped generating a 5′-ppp-5′-triphosphatelinkage between a terminal guanosine cap residue and the 5′-terminaltranscribed sense nucleotide of the mRNA molecule. This 5′-guanylate capmay then be methylated to generate an N7-methyl-guanylate residue. Theribose sugars of the terminal and/or anteterminal transcribednucleotides of the 5′ end of the mRNA may optionally also be2′-O-methylated. 5′-decapping through hydrolysis and cleavage of theguanylate cap structure may target a nucleic acid molecule, such as anmRNA molecule, for degradation. Modifications to the polynucleotides,primary constructs, and mmRNA of the present disclosure may generate anon-hydrolyzable cap structure preventing decapping and thus increasingmRNA half-life. Because cap structure hydrolysis requires cleavage of5′-ppp-5′ phosphorodiester linkages, modified nucleotides may be usedduring the capping reaction. For example, a Vaccinia Capping Enzyme fromNew England Biolabs (Ipswich, Mass.) may be used with.alpha.-thio-guanosine nucleotides according to the manufacturer'sinstructions to create a phosphorothioate linkage in the 5′-ppp-5′ cap.Additional modified guanosine nucleotides may be used such as.alpha.-methyl-phosphonate and seleno-phosphate nucleotides. Additionalmodifications include, but are not limited to, 2′-O-methylation of theribose sugars of 5′-terminal and/or 5′-anteterminal nucleotides of themRNA (as mentioned above) on the 2′-hydroxyl group of the sugar ring.Multiple distinct 5′-cap structures can be used to generate the 5′-capof a nucleic acid molecule, such as an mRNA molecule.

Flanking Regions Untranslated Regions (UTRs)

Untranslated regions (UTRs) of a gene are transcribed but nottranslated. The 5′UTR starts at the transcription start site andcontinues to the start codon but does not include the start codon,whereas the 3′UTR starts immediately following the stop codon andcontinues until the transcriptional termination signal. UTRs can beincorporated into the polynucleotides, primary constructs and/or mRNA ofthe present disclosure to enhance the stability of the molecule. UTRsalso be incorporated to ensure controlled down-regulation of thetranscript in case they are misdirected to undesired organs sites.

5′ UTR and Translation Initiation

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of the polynucleotides, primary constructs or mmRNAof the disclosure. For example, introns or portions of introns sequencesmay be incorporated into the flanking regions of the polynucleotides,primary constructs or mmRNA of the disclosure.

AU Rich Elements

AU rich elements (AREs) can be separated into three classes (Chen etal., Mol. Cell. Biol. 15:5777-5788, 1995; Chen et al., Mol. Cell Biol.15:2010-2018, 1995): Class I AREs contain several dispersed copies of anAUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs.Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) (SEQ IDNO: 38)nonamers. Molecules containing this type of AREs include GM-CSFand TNF-a. Class III ARES are less well defined. These U rich regions donot contain an AUUUA motif c-Jun and Myogenin are two well-studiedexamples of this class. Most proteins binding to the AREs are known todestabilize the messenger, whereas members of the ELAV family, mostnotably HuR, have been documented to increase the stability of mRNA. HuRbinds to AREs of all the three classes. Engineering the HuR specificbinding sites into the 3′ UTR of nucleic acid molecules will lead to HuRbinding and thus, stabilization of the message in vivo. Introduction,removal or modification of 3′ UTR AU rich elements (AREs) can be used tomodulate the stability of polynucleotides, primary constructs or mmRNAof the disclosure. When engineering specific polynucleotides, primaryconstructs or mmRNA, one or more copies of an ARE can be introduced tomake polynucleotides, primary constructs or mmRNA of the disclosure lessstable and thereby curtail translation and decrease production of theresultant protein. Likewise, AREs can be identified and removed ormutated to increase the intracellular stability and thus increasetranslation and production of the resultant protein. Transfectionexperiments can be conducted in relevant cell lines, usingpolynucleotides, primary constructs or mmRNA of the disclosure andprotein production can be assayed at various time pointspost-transfection. For example, cells can be transfected with differentARE-engineering molecules and by using an ELISA kit to the relevantprotein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48hour, and 7 days post-transfection. The polynucleotide, primaryconstruct, and mRNA of the disclosure can be formulated using naturaland/or synthetic polymers. Non-limiting examples of polymers which maybe used for delivery include, but are not limited to, DYNAMICPOLYCONJUGATE® (Arrowhead Research Corp., Pasadena, Calif.) formulationsfrom MIRUS® Bio (Madison, Wis.) and Roche Madison (Madison, Wis.),PHASERX™ polymer formulations such as, without limitation, SMARTTPOLYMER TECHNOLOGY™ (PHASERX®, Seattle, Wash.), DMRI/DOPE, poloxamer,VAXFECTIN® adjuvant from Vical (San Diego, Calif.), chitosan,cyclodextrin from Calando Pharmaceuticals (Pasadena, Calif.), dendrimersand poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, Calif.) and pH responsive co-blockpolymers such as, but not limited to, PHASERX® (Seattle, Wash.). Many ofthese polymer approaches have demonstrated efficacy in deliveringoligonucleotides in vivo into the cell cytoplasm (reviewed indeFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated byreference in its entirety). Two polymer approaches that have yieldedrobust in vivo delivery of nucleic acids, in this case with smallinterfering RNA (siRNA), are dynamic polyconjugates andcyclodextrin-based nanoparticles. The first of these delivery approachesuses dynamic polyconjugates and has been shown in vivo in mice toeffectively deliver siRNA and silence endogenous target mRNA inhepatocytes (Rozema et al., Proc Natl Acad Sci USA. 2007104:12982-12887; herein incorporated by reference in its entirety). Thisparticular approach is a multicomponent polymer system whose keyfeatures include a membrane-active polymer to which nucleic acid, inthis case siRNA, is covalently coupled via a disulfide bond and whereboth PEG (for charge masking) and N-acetylgalactosamine (for hepatocytetargeting) groups are linked via pH-sensitive bonds (Rozema et al., ProcNatl Acad Sci USA. 2007 104:12982-12887; herein incorporated byreference in its entirety). On binding to the hepatocyte and entry intothe endosome, the polymer complex disassembles in the low-pHenvironment, with the polymer exposing its positive charge, leading toendosomal escape and cytoplasmic release of the siRNA from the polymer.Through replacement of the N-acetylgalactosamine group with a mannosegroup, it was shown one could alter targeting from asialoglycoproteinreceptor-expressing hepatocytes to sinusoidal endothelium and Kupffercells. Another polymer approach involves using transferrin-targetedcyclodextrin-containing polycation nanoparticles. These nanoparticleshave demonstrated targeted silencing of the EWS-FLI1 gene product intransferrin receptor-expressing Ewing's sarcoma tumor cells(Hu-Lieskovan et al., Cancer Res. 2005 65: 8984-8982; hereinincorporated by reference in its entirety) and siRNA formulated in thesenanoparticles was well tolerated in non-human primates (Heidel et al.,Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated byreference in its entirety). Both of these delivery strategiesincorporate rational approaches using both targeted delivery andendosomal escape mechanisms.

Example 16: Restoration of Hearing in Otof^(−/−) Mice by theTrans-Splicing Approach

An experiment was performed to determine whether hearing could berestored in Otof^(−/−) mice using a combination of two different vectorsusing the methods described herein.

Materials and Methods

Cloning

Mouse otoferlin cDNA transcript variant 4 (KX060996; coding DNA sequence(CDS) identical to reference sequence NM_001313767) that had beensubcloned from cochlear cDNA (Strenzke et al. (2016) EMBO J. 352519-2535) was subcloned into the backbone for AAV production usingstandard cloning strategies including restriction digests and ligation.Both vectors contain ITRs of serotype 2. A CMV enhancer and humanJ-actin promoter were subcloned into the 5′ vector, which contains eGFPcDNA and a P2A signal (FIG. 1 ). The otoferlin CDS was split at theexon21-exon22 junction into two halves of about similar size. The 5′vector encodes the N-terminal part of otoferlin from amino acid 1 to844, the 3′ vector contains the coding sequence for amino acids 845 to1977 and woodchuck hepatitis virus posttranscriptional regulatoryelement (WPRE) and poly-adenlyation signals.

For the trans-splicing approach, a splice donor site (Trapani et al.(2014) EMBO Mol. Med. 6(2):194-211, 2014) follows the coding sequence inthe 5′ vector. In the 3′ vector, a splice acceptor site was subclonedjust before the coding sequence for otoferlin. Within the codingsequence, a silent mutation was introduced, generating an additionalsite for restriction digestion.

For the hybrid approach, an F1 phage recombinogenic region forhomologous recombination was subcloned right after the splice donor sitein the 5′ vector and before the splice acceptor site of the 3′ vector(FIG. 2 ). The plasmid identities were verified by Sanger sequencing.

Virus Production and Purification

Dual AAV vectors were produced by transient transfection of HEK293 cellsgrown in multi-level cell factories. The cells were co-transduced withhelper plasmids for virus production encoding serotype 6 capsidproteins. Purification of cell lysates was performed by iodixanoldensity-gradient ultracentrifugation, followed by a second purificationand concentration step by FPLC affinity-chromatography (Asai et al.(2015) Nat. Neurosci. 18 1584-1593; Tereshchenko et al. (2014)Neurobiol. Dis. 65 35-42). For the trans-splicing approach, the 5′vector achieved a concentration of ˜2.8×10⁸ transducing units/μL. The 3′vector reached ˜1.4×10⁸ transducing units/μL. For the hybrid approach,both viruses were purified simultaneously in the same solutions,reaching slightly higher virus titers.

Mouse Strains

Otoferlin knock-out (Otof^(−/−)) mice were generated as described(Reisinger et al. (2011) J. Neurosci. 31 4886-4895) and backcrossed forat least 5 generations to either C57/B16N or to CD1 strains. For virusinjection, F1 offspring from Otof^(−/−) CD1 females and Otof^(−/−)C57/B16N males were used. For wild type controls, Otof^(−/−) mice fromOtof^(+/−) C57/B16N breeding were crossed with CD1 wild type mice(Charles River).

Virus Injection

Mice at postnatal day 6 to 7 (P6-7) were anesthetized using 2.5%-5%isoflurane. The skin behind the left bulla was opened and the roundwindow niche was exposed. Virus solution in a glass capillary wasinjected through the round window membrane using a PLI-100A BASICPICOLITER microinjector (Harvard Apparatus GmbH, Germany) as pressuresource, thereby injecting about 0.2-0.5 μL solution per inner ear (Junget al. (2015) EMBO J. 34 2686-2702). The skin was closed and the pupswere raised by their mothers.

Immunohistochemistry

Immunostaining was performed as previously described (Strenzke et al.(2016) EMBO J. 35 2519-2535). Temporal bones were isolated afterdecapitation and a part of the cochlear bony shelf was opened and theround window was perforated. The temporal bones were fixed in 4%formaldehyde in PBS for 45 min at 4° C. Cochleas of mice older than P20were decalcified either for 5 min in Morse's solution or 2 days in 0.12MEDTA solution. Blocking was performed with 17% normal goat serum, 0.3%Triton X-100, 0.45 mM NaCl, and 20 mM phosphate buffer, pH 7.4.Antibodies were diluted in blocking solution and applied to the organ ofCorti situated in the temporal bones before apical and basal turns wereexcised. The following antibodies were used: goat IgG1 anti-Ctbp2 mouseanti-otoferlin (RRID:AB_881807, Abcam, Cambridge, UK, 1:300), rabbitanti-otoferlin (Synaptic Systems, Gottingen, Germany, 1:100), goatanti-calbindin D28k and secondary Alexa Fluor®405, Alexa Fluor®488-,Alexa Fluor®568-, Alexa Fluor®594-, and AlexaFluor647-labeled antibodies(Invitrogen, 1:200). Confocal microscopy images were acquired as stacksof 2D images with a step size of 0.6 μm using a laser scanning confocalmicroscope (Leica TCS SP5, Leica Microsystems CMS GmbH, Mannheim,Germany) with a 63×glycerol immersion objective (NA=1.456). The numberof synapses in 14-16 day old inner hair cells (IHCs) were counted usingthe cell counter plugin in ImageJ software as number of Ctbp2 spots.Image analysis to determine fractional levels of membrane boundotoferlin is described in Strenzke et al. (2016a) EMBO J. 35 2519-2535).

Electrophysiology

Ca²⁺ currents and plasma membrane capacitance from IHCs were measured bypatch-clamp of IHCs from the apical coils of freshly dissected organs ofCorti in the perforated-patch configuration at room temperature (20-25°C.) as described (Moser & Beutner (2000) Proc Natl Acad Sci USA 97883-888). The pipette solution contained 130 mM Cs-gluconate, 10 mMtetraethylammonium-chloride (TEA-Cl), 10 mM 4-aminopyridine (Merck,Darmstadt, Germany), 1 mM MgCl₂, 10 mM Cs-HEPES (pH 7.17, osmolarityapprox. 290 mOsm), 300 μg/mL amphotericin B (Calbiochem, La Jolla,Calif.). The extracellular solution contained 110 mM NaCl, 35 mM TEA-Cl,2.8 mM KCl, 2 mM CaCl₂), 1 mM MgCl₂, 10 mM Na-HEPES, 1 mM CsCl, 11.1 mMD-glucose (pH 7.2, osmolarity approx. 300 mOsm). Unless statedotherwise, all chemicals were obtained from Sigma-Aldrich (Taufkirchen,Germany). An EPC-9 amplifier (HEKA Electronics, Lambrecht, Germany)controlled by Pulse software was used to sample and filter currents at20 kHz and at 5 kHz, respectively. Potentials were corrected for liquidjunction potentials (−14 mV). ΔCm was measured using depolarizations ofdifferent durations to peak Ca²⁺ current potential, with 30-60 secinter-stimulus intervals, as previously described (Beutner & Moser(2000) Proc Natl Acad Sci USA 97 883-888). All currents wereleak-corrected using a P/6-protocol. The vesicle replenishment rate wascalculated as ΔCm during 100 ms depolarization minus ΔCm at 20 msdepolarization, divided by 45 aF per vesicle (Neef et al. (2007) TheJournal of Neuroscience 27 12933-12944) to get the number of vesicles.The number of active zones per cell was determined withimmunohistochemistry (10 synapses for transduced Otof^(−/−) IHCs, 14synapses for Otof^(+/+) IHCs).

ABR Recordings

Auditory brainstem responses were recorded as described (Jing et al.(2013) J Neurosci 33 4456-4467).

RNA Isolation, Reverse Transcription, PCR and Sequencing

Total RNA was isolated from acutely dissected organs of Corti withInvitrogen™ TRIzol™ Plus RNA Purification Kit (Thermo Fisher Scientific,12183555) according to the manufacturer's instructions and used as atemplate for cDNA synthesis using SuperScript® IV First-Strand SynthesisSystem (ThermoFisher Scientific, 18091050) with Oligo(dT)₂₀ and RandomHexamer primers. The cDNA was further used in PCR amplificationreactions using DreamTaq® Polymerase (Thermo Fisher Scientific, EP0702),using the following primers:

N-terminal fragment (SEQ ID NO: 24) 3′-CCCACAAGGCCAACGAGACGGATGAGGAC-5′and (SEQ ID NO: 25) 3′- AAGAGGCTTCGGGCCTGATACATGTGTGCT-5′;Assembly fragment (SEQ ID NO: 26) 3′-ACGGCAATGAAGTCGATGGTATGTCCCGGC-5′;C-terminal fragment (SEQ ID NO: 27) 3′-CTGACCTGCCACCCATCAATGGCCCAGTGG-5′and (SEQ ID NO: 28) 3′-CTATGCGCTCCTCCTCTGTGGAGCCATCCT-5′.

All bands were excised, cloned into a pCR2.1™-TOPO® vector using theTOPO® TA® Cloning Kit (Thermo Fisher Scientific, 450641), and shot intoOne Shot™ TOP10 Electrocomp™ E. coli cells (Thermo Fisher Scientific,C404050). All clones were screened for the correct insert and verifiedby Sanger sequencing.

Results

To test the trans-splicing approach, dual-AAV vectors were generated,each comprising one half of the otoferlin CDS. In the first vector, ahuman ß-actin promoter and a CMV enhancer were used to drive theexpression of eGFP and the 5′ otoferlin fragment, separated by a P2Asequence (FIG. 1 ). The second vector contained the 3′ otoferlin CDS andmRNA stabilizing elements. AAV vectors underwent head-to-tailmultimerization in the nuclei of target cells by non-homologous endjoining of the inverted terminal repeats (ITRs), thereby promoting theassembly of the two vector genomes. A splice donor site in the 5′ vectorand a splice acceptor site in the 3′ vector were included to force theprocessing of a correct full-length otoferlin mRNA (FIG. 1 ).

Otoferlin knock-out Otof^(−/−) animals on postnatal day 6-7 (P6-7) wereco-injected with both AAVs (˜1.4-2.8×10⁸ transducing units/μL) throughthe round window membrane of the left ear (FIG. 3 ). Non-injectedOtof^(−/−) littermates and wild-type animals, some of which wereinjected with eGFP-encoding AAVs, served as controls. At P23-28, hearingwas tested using auditory brainstem recordings (ABRs), and the innerhair cell (IHC) transduction rate and otoferlin expression levels weretested using immunohistochemistry. As shown in FIG. 3 , innon-transduced Otof^(−/−) mice, ABR recordings elicited a prominentsummating potential (SP) due to the depolarization of IHCs, but thesesignals were not transmitted to the brainstem, resulting in a flat line.After dual-AAV transduction, mice displayed typical ABR waves,indicating that auditory information was processed to the brain stem.

In response to click stimuli, thresholds in successfully transduced earswere at 54±3 dB (range: 30-70 dB). In control experiments,non-transduced Otof^(−/−) littermate animals showed no ABR waves exceptthe summating potential, even at 100 dB sound stimulus. Pure tonestimuli elicited ABR responses in most transduced ears, but not innon-transduced Otof^(−/−) littermate controls (FIGS. 3-6 ). Pure toneABRs were detectable for low (6 kHz), mid (12 kHz), and high frequencies(24 kHz). The Otof^(+/+) controls showed normal ABR wave forms with athreshold of 39±1 dB (range: 30-40 dB) (FIGS. 4 and 5 ). As shown inFIG. 4 , the amplitudes of ABR waves in transduced Otof^(−/−) mice aresmaller than in wild type Otof^(wt/wt) mice transduced with dual-AAVwith enhanced green fluorescent protein (eGFP).

To test for correct concatemerization and reassembly of the full-lengthotoferlin mRNA in the right orientation, mRNA was extracted fromtransduced and non-transduced P14 Otof^(−/−) organs of Corti. Afterreverse transcription into cDNA, three fragments from otoferlin mRNAwere amplified: one encoded by the 5′ virus, one encoded by the 3′virus, and one fragment covering the site of assembly of the twovectors. Sanger sequencing of the PCR amplicons revealed the correctotoferlin transcripts containing the silent mutation of virallytransduced otoferlin, indicating correct assembly of dual-AAV transducedotoferlin mRNA.

To study the inner hair cell (IHC) transduction rate and otoferlinexpression levels, organs of Corti were dissected at P18-28 and analyzedby immunohistochemistry using two anti-otoferlin antibodies, one bindingto the N-terminal part and the other binding to the very C-terminal partof otoferlin (FIG. 6 and FIG. 7 ). As shown in FIG. 7 , calbindin wasused as marker for inner and outer hair cells. Cells expressing eGFPindicated virus transduction of at least the 5′ virus. Anti-otoferlinantibody staining (Abcam) in the N-terminal half of otoferlin indicatedexpression of the N-terminal part of otoferlin. C-terminalanti-otoferlin antibody staining (Synaptic Systems) indicated expressionof the C-terminal part of otoferlin. Taken together, all three panels inFIG. 7 demonstrated that full-length otoferlin was expressed in IHCs.Twelve to fifty-one percent (on average 32±4%, s.e.m.) of IHCs showedfull-length otoferlin expression with higher IHC transduction rates inthe apex of the cochlea (low frequency region, 35±5% transduction rate;range: 19-52%) than in the basal turn (24±6%, range: 0-51%). About 10%of all IHCs showed N-terminal otoferlin signal, but no C-terminalotoferlin expression, presumably indicating transduction of the 5′vector only. No signal was observed in the Otof^(−/−) controllittermates. Remarkably, both N-terminal and C-terminal otoferlin signalwas found only in IHCs, whereas eGFP fluorescence could additionally befound in outer hair cells (OHCs) as well as in other cell types that didnot express otoferlin in parallel. Thus, despite AAV transduced inseveral cell types in the organ of Corti, expression of otoferlin wasrestricted to IHCs. To quantify otoferlin expression levels,immunofluorescence using the N-terminal anti-otoferlin antibody inconfocal stacks of immunostained IHCs was measured (as in Strenzke etal. (2016) EMBO J. 35 2519-2535). In dual-AAV transduced Otof^(−/−)IHCs, otoferlin levels were found to be ˜30% of wild-type controls.

Since synapses in Otof^(−/−) mice are partially lost during the secondpostnatal week, an experiment was conducted to determine whetherexpression of otoferlin in these IHCs affected synapse numbers. Synapticribbons were immunolabelled; 9±0.3 synapses were found in 26 day oldnon-transduced Otof^(−/−) IHCs (n=42 cells, N=3 animals). In dual-AAVtransduced Otof^(−/−), 10±0.2 synapses were identified by eGFPfluorescence (n=59 cells; N=3 animals). In contrast, wild-type IHCsdisplayed 13-16 synapses per IHC (Strenzke et al. (2016) EMBO J. 352519-2535). Thus, the expression of otoferlin after dual-AAV injectionat P6 partially prevented the loss of synapses from P26 Otof^(−/−) IHCs.

The data in FIGS. 15 and 16 also show that the hybrid approach(administration of the vectors shown in FIG. 2 ) also restores otoferlinactivity in the IHCs of Otof-mice.

These data indicate that the presently claimed methods can restorehearing in mammals having an inactivating mutation in an otoferlin gene.

Example 17: Restoration of Exocytosis in Response to ShortDepolarization Stimuli Restored by Trans-Splicing Dual-AAV MediatedExpression of Otoferlin in IHCs of Otof^(−/−) Mice

An experiment was performed to determine whether the presently claimedmethods would restore fusion of synaptic vesicles with inner auditoryhair cells in Otof^(−/−) mice.

Since the fusion of synaptic vesicles increases the plasma membranesurface of the cell, this fusion can be measured as an increase inplasma membrane capacitance (ΔCm). In order to quantify exocytosis ofthe readily releasable pool of vesicles and sustained exocytosis, thechange of plasma membrane capacitance (ΔC_(m)) was measured inperforated patch-clamp configuration (FIGS. 8-10 ). Exocytosis wasrecorded as the change in membrane capacitance after depolarizing theIHC to −14 mV for the indicated duration.

IHCs from acutely explanted organs of Corti of mice were depolarized atpostnatal days 14 to 18 (P14-P18) to the voltage where maximum Ca²⁺currents were elicited, typically −14 mV. Ca²⁺ currents and ΔC_(m) fromOtof^(−/−) IHCs with and without viral transduction were recorded, withvirally transduced IHCs being identified by their eGFP fluorescenceduring the experiment. Consistent with the synapse numbers in untreatedand transduced Otof^(−/−) IHCs, Ca²⁺ currents were found to becomparable in size to those of Otof^(+/+) age- and background matchedcontrols (FIGS. 8 and 10 ). As shown in FIG. 8 , Ca²⁺ currents did notdiffer in amplitude or gating properties between non-transducedOtof^(−/−) IHCs, dual-AAV transduced IHCs, or wild-type IHCs frombackground-matched control animals. In response to depolarization pulsesof 5 or 10 ms, exocytosis in transduced Otof^(−/−) IHCs was found to beof similar size as in Otof^(+/+) IHCs, while almost no change in C_(m)could be detected in non-transduced Otof^(−/−) IHCs (FIG. 9 ). Indeed,for short depolarization stimuli up to 20 ms, exocytosis was wild-typelike in dual-AAV transduced IHCs of Otof^(−/−) mice, indicating anintact, readily-releasable pool of vesicles. During sustaineddepolarization, vesicles of the readily-releasable pool need to bereplenished; otoferlin is known to be required for this process(Pangrsic et al. (2010) Nat. Neurosci. 13 869-876; Strenzke et al.(2016) EMBO J. 35 2519-2535). For longer depolarization stimuli,exocytosis was in the range of the mildly hearing impairedOtof^(I515T/I515T) mice (Strenzke et al. (2016) EMBO J. 35 2519-2535).As shown in FIG. 9 , for 50-100 ms depolarizations, exocytosis intransduced Otof^(−/−) IHCs was found to be ˜60% of Otof^(+/+) IHCs,while non-transduced Otof^(−/−) IHCs showed hardly any vesicle fusion,as previously described (Roux et al. (2006) Cell 127 277-289). The ratesof vesicle replenishment were calculated in individual transduced cells.Approximately three hundred and eighty vesicles/sec/active zone werefound to undergo exocytosis, compared to 750 vesicles/sec/active zone inOtof^(+/+) IHCs (Strenzke et al. (2016) EMBO J. 35 2519-2535). Thus, intransduced Otof^(−/−) IHCs, the rate of vesicle replenishment to the RRPcould be partially recovered and was between that of wild type controlsand mildly hearing impaired OtofI515T^(/1515T) mice (Strenzke et al.(2016) EMBO J. 35 2519-2535). As shown in FIG. 10 , Ca integrals duringthe depolarization step indicate that a similar charge of Ca²⁺ enteredIHCs in transduced and non-transduced Otof^(−/−) IHCs and in wild-typecontrol cells.

The AAV serotype 6 was chosen, which resulted in IHC transduction ratesof up to 51%. The artificial serotype Anc80L65 might, as recently hasbeen shown (Landegger et al. (2017) Nat. Biotechnol. 35 280-284),increase the IHC transduction rate further, especially when applied tomore mature ears (Suzuki et al. (2017) Scientific Reports 7 45524).

In sum, these data show that hearing was restored in nine injectedOtof−/− animals, using the methods described herein. These data alsoindicate that hearing thresholds get better the more IHCs expressotoferlin.

Example 18: Adeno-Associated Virus (AAV) Trans-Splicing Strategy

At least two different nucleic acid vectors (e.g., AAV vectors) can beused to reconstitute an active otoferlin gene (e.g., a full-lengthotoferlin gene) within a cell following intermolecular concatamerizationand trans-splicing. See, e.g., Yan et al., Proc. Natl. Acad. Sci. U.S.A.97:12; 6716-6721, 2000, incorporated in its entirety herein.

In some examples, two different nucleic acid vectors will be used. Afirst nucleic acid vector can include a promoter (e.g., any of thepromoters described herein), a first coding sequence that encodes anN-terminal portion of an otoferlin protein positioned 3′ of the promoter(e.g., any of the sizes of a portion of an otoferlin protein describedherein and/or any of the N-terminal portions of an otoferlin proteindescribed herein), and a splicing donor signal sequence positioned atthe 3′ end of the first coding sequence. A second nucleic acid vectorcan include a splicing acceptor signal sequence, a second codingsequence that encodes a C-terminal portion of an otoferlin protein(i.e., the entire portion of the otoferlin protein that is not includedin the N-terminal portion) positioned at the 3′ end of the splicingacceptor signal sequence (e.g., any of the sizes of a portion of anotoferlin protein described herein and/or any of the C-terminal portionsof an otoferlin protein described herein), and a polyadenylationsequence at the 3′ end of the second coding sequence (e.g., any of thepolyadenylation sequences described herein). In some embodiments, eachof the encoded portions is at least 30 amino acid residues in length(e.g., at least 50 amino acids, at least 75 amino acids, or at least 100amino acids in length), the amino acid sequence of each of the encodedportions does not overlap with the sequence of the other encodedportion, and no single vector of the two different vectors encodes anactive otoferlin protein (e.g., a full-length otoferlin protein). Whenthe two coding sequences of the two vectors are expressed in a mammaliancell (e.g., any of the mammalian cells described herein), splicingoccurs between the splicing donor signal sequence and the splicingacceptor signal sequence, thereby forming a recombined mRNA that encodesan active otoferlin protein (e.g., a full-length otoferlin protein).

In another example, three different nucleic acid vectors can be used. Afirst nucleic acid vector can include a portion of a promoter sequence(e.g., any of the promoter sequences described herein), a first codingsequence of an otoferlin gene that encodes a first portion of anotoferlin protein (e.g., any of the otoferlin coding sequences describedherein) positioned 3′ of the promoter, and a first splicing donor signalsequence positioned at the 3′ end of the first coding sequence. A secondnucleic acid vector can include a first splicing acceptor signalsequence, a second coding sequence of an otoferlin gene that encodes asecond portion of an otoferlin protein positioned at the 3′ end of thefirst splicing acceptor signal sequence, and a second splicing donorsignal sequence positioned at the 3′ end of the second coding sequence(e.g., any of the splicing donor signals described herein). A feature ofthe second nucleic acid vector will be that self-splicing cannot occur(i.e., splicing will not occur between the second splicing donor signalsequence and the first splicing acceptor signal sequence of the secondnucleic acid vector). In some embodiments, the splicing donor signalsequence of the first nucleic acid vector and the second splicing donorsignal of the second nucleic acid vector are the same (e.g., any of thesplicing donor signals described herein or known in the art). In someembodiments, the first splicing donor signal sequence of the firstnucleic acid vector and the second splicing donor signal sequence of thesecond nucleic acid vector are different (e.g., any of the splicingdonor signal sequences described herein or known in the art). A thirdnucleic acid vector will include a second splicing acceptor signalsequence, a third coding sequence of an otoferlin gene that encodes athird portion of an otoferlin protein positioned at the 3′ end of thesecond splicing acceptor signal sequence, and a polyadenylation sequencepositioned at the 3′ end of the third coding sequence (e.g., any of thepolyadenylation sequences described herein). In such methods where threenucleic acid vectors are used, the first splicing donor sequence and thefirst splicing acceptor sequence can assemble together and the secondslicing donor sequence and the second slicing acceptor sequence canassemble together (recombine), and the portions of otoferlin proteinrespectively encoded by the first, second, and third coding sequences donot overlap, and when introduced into a mammalian cell (e.g., any of themammalian cells described herein), splicing occurs between the firstsplicing donor sequence and the first splicing acceptor sequence, andbetween the second splicing donor sequence and the second splicingacceptor sequence, to form a spliced nucleic acid that encodes an activeotoferlin protein (e.g., a full-length otoferlin protein). Based on thestrategies provided above, one skilled in the art would understand howto develop a strategy using four, five, or six different nucleic acidvectors.

In any of the examples of these methods, the amino acid sequence of eachof the encoded portions does not overlap with the sequence any of theother encoded portions, and no single vector encodes an active otoferlinprotein (e.g., a full-length otoferlin protein).

Each of the at least two different vectors includes a coding sequencethat encodes a different portion of an otoferlin protein, each of theencoded portions can be at least 30 amino acids (e.g., between about 30amino acids to about 1600 amino acids, or any of the other subranges ofthis range described herein).

In some embodiments, each of the coding sequences can include at leastone exon and at least one intron of SEQ ID NO: 12 (e.g., at least twoexons and at least one intron, at least two exons and at least twointrons, at least three exons and at least one intron, at least threeexons and at least two introns, or at least three exons and at leastthree introns). In some embodiments, each of the at least two differentvectors includes a coding sequence that encodes a different portion ofan otoferlin protein, each of the encoded portions can encode up to 80%of the amino acid sequence of SEQ ID NO: 5 (e.g., up to 10%, up to 20%,up to 30%, up to 40%, up to 50%, up to 60%, or up to 70% of SEQ ID NO:5) such that each of the encoded portions is non-overlapping. In someembodiments, each of the at least two different vectors includes acoding sequence that encodes a different portion of an otoferlinprotein, each of the encoded portions encoding up to 80% of the aminoacid sequence of SEQ ID NO: 5 (e.g., up to 10%, up to 20%, up to 30%, upto 40%, up to 50%, up to 60%, or up to 70% of SEQ ID NO: 5), such thateach of the encoded portions is non-overlapping.

Each of the at least two nucleic acid vectors may further include aninverted terminal repeat (ITR) to allow head-to-tail recombination. TheITR will be subsequently removed via splicing. For example, the ITRcould be a palindromic double-D ITR as described in Yan et al., Proc.Natl. Acad. Sci. U.S.A. 97(12):6716-6721, 2000, incorporated in itsentirety herein. For example, the ITR could be a AAV serotype-2 ITR asdescribed in Gosh et al., Mol. Ther. 16:124-130, 2008, and Gosh et al.,Human Gene Ther. 22: 77-83, 2011. Non-limiting examples of splicingacceptor and/or donor signal sequences are known in the art. See, e.g.,Reich et al., Human Gene Ther. 14(1):37-44, 2003, and Lai et al. (2005)Nat. Biotechnol. 23(11):1435-1439, 2005, 2005. The splicing donor andacceptor signal sequences can be any endogenous intron splicing signalof a gene (e.g., an otoferlin gene).

For example, the splicing donor signal sequence can be: (SEQ ID NO: 64)5′-GTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCT TGCGTTTCT-3′and the splicing acceptor signal can be: (SEQ ID NO: 110)5′-ATAGGCACCTATTGGTCTTACTGACATCCACTTTG CCTTTCTCTCCACAG-3′(see, e.g., Trapani et al., EMBO Mol. Med. 6(2):194-211, 2014).

Methods of evaluating splicing and splicing efficiency are known in theart (see, e.g., Lai et al., Nat. Biotechnol. 23(11): 1435-1439, 2005).

Example 19: Hybrid Vector Trans-Splicing Strategy Using an AlkalinePhosphatase (AP) Highly Recombinogenic Exogenous Gene Region

At least two (e.g., two, three, four, five, or six) different nucleicacid vectors (e.g., AAV vectors) can also be used in any of the methodsdescribed herein to reconstitute an active otoferlin gene (e.g., afull-length otoferlin gene) within a cell following intermolecularconcatamerization, marker gene-mediated recombination, andtrans-splicing. This strategy is a hybrid strategy as it will includehomologous recombination and/or trans-splicing. See, e.g., Gosh et al.,Mol. Ther. 16: 124-130, 2008; Gosh et al., Human Gene Ther. 22: 77-83,2011; and Duan et al., Mol. Ther. 4: 383-391, 2001, each incorporated inits entirety herein. As used herein, a detectable marker gene can be ahighly recombinogenic DNA sequence that will allow for codingsequence-independent recombination. An non-limiting example of adetectable marker gene is an alkaline phosphatase (AP) gene. Forexample, the detectable marker gene can be the middle one-third of thehuman placental AP complementary DNA, which is 872 bp in length (see,e.g., Gosh et al., 2008). At least two different nucleic acid vectorswill contain a detectable marker gene (e.g., any of the detectablemarker genes described herein). Since the hybrid vector will beconstructed based on a trans-splicing vector as described in Example 18,an active otoferlin gene (e.g., a full-length otoferlin gene) may bereconstituted using either ITR-mediated recombination and trans-splicingor detectable marker gene-mediated (e.g., AP-gene mediated)recombination and trans-splicing. After trans-splicing, an activeotoferlin gene (e.g., a full-length otoferlin gene) will bereconstituted in the genomic DNA of a mammalian cell (e.g., anymammalian cell described herein).

In one example, two different nucleic acid vectors will be used. A firstnucleic acid vector can include a promoter (e.g., any of the promotersdescribed herein), a first coding sequence that encodes an N-terminalportion of an otoferlin protein positioned 3′ of the promoter (e.g., anyof the sizes of a portion of an otoferlin protein described hereinand/or any of the N-terminal portions of an otoferlin protein describedherein), a splicing donor signal sequence positioned at the 3′ end ofthe first coding sequence, and a first detectable marker gene positioned3′ of the splicing donor signal sequence. A second nucleic acid vectorcan include a second detectable marker gene, a splicing acceptor signalsequence positioned 3′ of the second detectable marker gene, a secondcoding sequence that encodes a C-terminal portion of an otoferlinprotein positioned at the 3′ end of the splicing acceptor signalsequence (e.g., any of the sizes of a portion of an otoferlin proteindescribed herein and/or any of the C-terminal portions of an otoferlinprotein described herein), and a polyadenylation sequence at the 3′ endof the second coding sequence (e.g., any of the polyadenylationsequences described herein). In some embodiments, each of the encodedportions is at least 30 amino acid residues in length (e.g., at least 50amino acids, at least 75 amino acids, or at least 100 amino acids inlength), the amino acid sequence of each of the encoded portions do notoverlap, and no single vector of the two different vectors encodes anactive otoferlin protein (e.g., a full-length otoferlin protein). Whenintroduced into a mammalian cell (e.g., any of the mammalian cellsdescribed herein) splicing occurs between the splicing donor signalsequence and the splicing acceptor signal sequence, thereby forming anRNA acid that encodes an active otoferlin protein (e.g., a full-lengthotoferlin protein).

In another example, three different nucleic acid vectors can be used. Afirst nucleic acid vector can include a portion of promoter sequence(e.g., any of the promoter sequences described herein), a first codingsequence of an otoferlin gene that encodes a first portion of anotoferlin protein (e.g., any of the otoferlin coding sequences describedherein) positioned 3′ of the promoter, a first splicing donor signalsequence positioned at the 3′ end of the first coding sequence, and afirst detectable marker gene. A second nucleic acid vector can include asecond detectable marker gene, a first splicing acceptor signal sequencepositioned 3′ of the second detectable marker gene, a second codingsequence of an otoferlin gene that encodes a second portion of anotoferlin protein positioned at the 3′ end of the first splicingacceptor signal sequence, a second splicing donor signal sequencepositioned at the 3′ end of the second coding sequence (e.g., any of thesplicing donor signals described herein), and a third detectable markergene. A feature of the second nucleic acid vector will be thatself-splicing cannot occur (i.e., splicing will not occur between thesecond splicing donor signal sequence and the first splicing acceptorsignal sequence of the second nucleic acid vector). In some embodiments,the splicing donor signal sequence of the first nucleic acid vector andthe second splicing donor signal of the second nucleic acid vector arethe same (e.g., any of the splicing donor signals described herein orknown in the art). In some embodiments, the first splicing donor signalsequence of the first nucleic acid vector and the second splicing donorsignal sequence of the second nucleic acid vector are different (e.g.,any of the splicing donor signal sequences described herein or known inthe art). A third nucleic acid vector can include a fourth detectablemarker gene, a second splicing acceptor signal sequence positioned 3′ ofthe fourth detectable marker gene, a third coding sequence of anotoferlin gene that encodes a third portion of an otoferlin proteinpositioned at the 3′ end of the second splicing acceptor signalsequence, and a polyadenylation sequence positioned at the 3′ end of thethird coding sequence (e.g., any of the polyadenylation sequencesdescribed herein). In such methods where three nucleic acid vectors areused, the first splicing donor sequence and the first splicing acceptorsequence can assemble together (recombine) and the second slicing donorsequence and the second slicing acceptor sequence can assemble together(recombine), and the portion of otoferlin protein encoded by the first,second, and third coding sequences do not overlap, and when introducedinto a mammalian cell (e.g., any of the mammalian cells describedherein), splicing occurs between the first splicing donor sequence andthe first splicing acceptor sequence, and between the second splicingdonor sequence and the second splicing acceptor sequence, to form arecombined nucleic acid that encodes an active otoferlin protein (e.g.,a full-length otoferlin protein). As can be appreciated in the art, whenthree nucleic acid vectors are used, two of the at least two differentnucleic acid vectors can include a detectable marker gene (e.g., an APmarker gene), and one of the at least two different nucleic acid vectorsmay include a splicing acceptor signal sequence that is complementary toa splicing donor signal sequence in a nucleic acid vector that includesa detectable marker gene. For example, in some embodiments, the firstand second nucleic acid vectors can include a detectable marker gene(e.g., an AP marker gene), and the third nucleic acid vector willinclude a splicing acceptor signal sequence that is complementary to thesplicing donor signal sequence in the second nucleic acid vector, andthe third nucleic acid vector will not include a detectable marker gene(e.g., an AP marker gene). In other examples, the second and thirdnucleic acid vector can include a detectable marker gene (e.g., an APmarker gene), and the first nucleic acid vector will include a splicingdonor signal sequence that is complementary to the splicing acceptorsignal sequence in the second nucleic acid vector and the first nucleicacid vector will not include a detectable marker gene (e.g., an APmarker gene).

Based on the strategies provided above, one skilled in the art wouldunderstand how to develop a strategy using four, five, or six vectors.

The coding sequences provided in the at least two nucleic acid vectors(e.g., two, three, four, five or six) will not be overlapping. Each ofthe at least two different vectors can include a coding sequence thatencodes a different portion of an otoferlin protein, each of the encodedportions being, e.g., at least 30 amino acids (e.g., about 30 aminoacids to about 1600 amino acids, or any of the other subranges of thisrange described herein).

In some embodiments, each of the at least two different vectors includesa coding sequence that encodes a different portion of an otoferlinprotein, each of the encoded portions encoding at least one exon and atleast one intron of SEQ ID NO: 12 (e.g., at least two exons and at leastone intron, at least two exons and at least two introns, at least threeexons at least one intron, at least three exons and at least twointrons, or at least three exons and at least three introns). In someembodiments, each of the at least two different vectors include a codingsequence that encodes a different portion of an otoferlin protein, eachof the encoded portions encoding up to 80% of SEQ ID NO: 5 (e.g., up to10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%, up to 70% ofSEQ ID NO: 5) such that each of the encoded portions is non-overlapping.In some embodiments, each of the at least two different vectors includea coding sequence that encodes a different portion of an otoferlinprotein, each of the encoded portions encoding up to 80% of SEQ ID NO: 5(e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%,or up to 70% of SEQ ID NO: 5), such that each of the encoded portions isnon-overlapping.

As described in Example 18, each of the at least two nucleic acidvectors may further include an inverted terminal repeat (ITR) to allowhead-to-tail recombination. The ITR will be subsequently removed viasplicing. Examples of ITRs and splicing acceptor and/or donor signalsequences are known in the art and have been described in Example 18.

Example 20: Hybrid Vector Trans-Splicing Strategy Using a F1 PhageHighly Recombinogenic Exogenous Gene Region (AK)

At least two (e.g., two, three, four, five, or six) different nucleicacid vectors (e.g., AAV vectors) can also be used in any of the methodsdescribed herein to reconstitute an active otoferlin gene (e.g., afull-length otoferlin gene) within a cell following intermolecularconcatamerization, marker gene-mediated recombination, andtrans-splicing. See, e.g., the vectors shown in FIG. 2 . This strategyis a hybrid strategy as it will include homologous recombination and/ortrans-splicing. See, e.g., Trapani et al., EMBO Mol. Med. 6(2):194-211,2014, incorporated in its entirety herein. As used herein, an F1 phagerecombinogenic region (AK) will be used to allow codingsequence-independent recombination. The F1 phage recombinogenic regionmay be a 77 bp recombinogenic region from the F1 phage genome asdescribed in Trapani et al. (2014) EMBO Mol. Med. 6(2):194-211, 2014. Atleast two different nucleic acid vectors will contain an F1 phagerecombinogenic region. Since the hybrid vector will be constructed basedon a trans-splicing vector as described in Example 18, a nucleic acidencoding an active otoferlin protein (e.g., a full-length stereocilinprotein) may be generated using either ITR-mediated recombination andtrans-splicing or F1 phage recombinogenic region-induced recombinationand trans-splicing. After trans-splicing, a nucleic acid encoding anactive otoferlin protein (e.g., a full-length otoferlin protein) will begenerated in a mammalian cell (e.g., any of the mammalian cellsdescribed herein).

In one example, two different nucleic acid vectors will be used. A firstnucleic acid vector can include a promoter (e.g., any of the promotersdescribed herein), a first coding sequence that encodes an N-terminalportion of an otoferlin protein positioned 3′ of the promoter (e.g., anyof the sizes of a portion of an otoferlin protein described hereinand/or any of the N-terminal portions of an otoferlin protein describedherein), a splicing donor signal sequence positioned at the 3′ end ofthe first coding sequence, and an F1 phage recombinogenic regionpositioned 3′ of the splicing donor signal sequence. A second nucleicacid vector can include an F1 phage recombinogenic region, a splicingacceptor signal sequence positioned 3′ of the F1 phage recombinogenicregion, a second coding sequence that encodes a C-terminal portion of anotoferlin protein positioned at the 3′ end of the splicing acceptorsignal sequence (e.g., any of the sizes of a portion of an otoferlinprotein described herein and/or any of the C-terminal portions of anotoferlin protein described herein), and a polyadenylation sequence atthe 3′ end of the second coding sequence (e.g., any of thepolyadenylation sequences described herein). In some embodiments, eachof the encoded portions is at least 30 amino acid residues in length(e.g., at least 50 amino acids, at least 75 amino acids, or at least 100amino acids in length), the amino acid sequence of each of the encodedportions do not overlap, and no single vector of the two differentvectors encodes an active otoferlin protein (e.g., a full-lengthotoferlin protein). When the vectors are introduced into a mammaliancell (e.g., any of the mammalian cells described herein), splicingoccurs between the splicing donor signal sequence and the splicingacceptor signal sequence, thereby forming a recombined nucleic acid thatencodes an active otoferlin protein (e.g., a full-length otoferlinprotein).

In another example, three different nucleic acid vectors will be used. Afirst nucleic acid vector can include a portion of promoter sequence(e.g., any of the promoter sequences described herein), a first codingsequence of an otoferlin gene that encodes a first portion of anotoferlin protein (e.g., any of the otoferlin coding sequences describedherein) positioned 5′ of the promoter, a first splicing donor signalsequence positioned at the 3′ end of the first coding sequence, and anF1 phage recombinogenic region. A second nucleic acid vector can includean F1 phage recombinogenic region, a first splicing acceptor signalsequence positioned 3′ of the F1 phage recombinogenic region, a secondcoding sequence of an otoferlin gene that encodes a second portion of anotoferlin protein positioned at the 3′ end of the first splicingacceptor signal sequence, a second splicing donor signal sequencepositioned at the 3′ end of the second coding sequence (e.g., any of thesplicing donor signals described herein), and an F1 phage recombinogenicregion. A feature of the second nucleic acid vector will be thatself-splicing cannot occur (i.e., splicing will not occur between thesecond splicing donor signal sequence and the first splicing acceptorsignal sequence of the second nucleic acid vector). In some embodiments,the splicing donor signal sequence of the first nucleic acid vector andthe second splicing donor signal of the second nucleic acid vector arethe same (e.g., any of the splicing donor signals described herein orknown in the art). In some embodiments, the first splicing donor signalsequence of the first nucleic acid vector and the second splicing donorsignal sequence of the second nucleic acid vector are different (e.g.,any of the splicing donor signal sequences described herein or known inthe art). A third nucleic acid vector can include an F1 phagerecombinogenic region, a second splicing acceptor signal sequencepositioned 3′ of the F1 phage recombinogenic region, a third codingsequence of an otoferlin gene that encodes a third portion of anotoferlin protein positioned at the 3′ end of the second splicingacceptor signal sequence, and a polyadenylation sequence positioned atthe 3′ end of the third coding sequence (e.g., any of thepolyadenylation sequences described herein). In such methods where threenucleic acid vectors are used, the first splicing donor sequence and thefirst splicing acceptor sequence can assemble together (recombine) andthe second slicing donor sequence and the second slicing acceptorsequence can assemble together (recombine), and the portion of otoferlinprotein encoded by the first, second, and third coding sequences do notoverlap, and when introduced into a mammalian cell (e.g., any of themammalian cells described herein), splicing occurs between the firstsplicing donor sequence and the first splicing acceptor sequence, andbetween the second splicing donor sequence and the second splicingacceptor sequence, to form a recombined nucleic acid that encodes anactive otoferlin protein (e.g., a full-length otoferlin protein). As canbe appreciated in the art when three nucleic acid vectors are used, twoof the different nucleic acid vectors can include an F1 phagerecombinogenic region and one of the different nucleic acid vectors mayinclude a splicing acceptor signal sequence that is complementary to asplicing donor signal sequence in a nucleic acid vector that includes anF1 phage recombinogenic region. For example, in some embodiments, thefirst and second nucleic acid vectors can include an F1 phagerecombinogenic region, and the third nucleic acid vector will include asplicing acceptor signal that is complementary to the splicing donorsignal sequence in the second nucleic acid vector, and the third nucleicacid vector will not include an F1 phage recombinogenic region (e.g., anAP marker gene). In other examples, the second and third nucleic acidvector can include an F1 phage recombinogenic region and the firstnucleic acid vector will include a splicing donor signal sequence thatis complementary to the splicing acceptor signal sequence in the secondnucleic acid vector and the first nucleic acid vector will not includean F1 phage recombinogenic region. Based on the strategies providedabove, one skilled in the art would understand how to develop a strategyusing four, five, or six vectors.

The coding sequences provided in each of the at least two nucleic acidvectors (e.g., two, three, four, five or six) will not be overlapping.Each of the at least two different vectors include a coding sequencethat encodes a different portion of an otoferlin protein, each of theencoded portions being at least 30 amino acids (e.g., about 30 aminoacids to about 1600 amino acids, or any of the subranges of this rangedescribed herein).

In some embodiments, each of the at least two different vectors includea coding sequence that encodes a different portion of an otoferlinprotein, each of the encoded portions encoding at least one exon and atleast one intron of SEQ ID NO: 12 (e.g., at least two exons and at leastone intron, at least two exons and at least two introns, at least threeexons at least one intron, at least three exons and at least twointrons, or at least three exons and at least three introns). In someembodiments, each of the at least two different vectors includes acoding sequence that encodes a different portion of an otoferlinprotein, each of the encoded portions encoding up to 80% of SEQ ID NO: 5(e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to 50%, up to 60%,or up to 70% of SEQ ID NO: 5) such that each of the encoded portions isnon-overlapping. In some embodiments, each of the at least two differentvectors include a coding sequence that encodes a different portion of anotoferlin protein, each of the encoded portions encoding up to 80% ofSEQ ID NO: 5 (e.g., up to 10%, up to 20%, up to 30%, up to 40%, up to50%, up to 60%, or up to 70% of SEQ ID NO: 5), such that each of theencoded portions is non-overlapping.

As described in Example 18 each of the at least two nucleic acid vectorsmay further include an inverted terminal repeat (ITR) to allowhead-to-tail recombination. The ITR will be subsequently removed viasplicing. Examples of ITRs and splicing acceptor and/or donor signalsare known in the art and have been described in Example 18.

These methods have been used to improve hearing in five animals. Thedata in FIGS. 15 and 16 show that the Otof^(−/−) mice administered thevectors shown in FIG. 2 restores otoferlin function in the IICs ofOtof^(−/−) mice.

Example 21: In Vitro Expression of Full-Length Human Otoferlin Using TwoVectors

A variety of different vectors were generated which each include anucleic acid sequence that encodes a portion of otoferlin: p109 (SEQ IDNO: 84), which is shown in FIG. 38 ; p105 (SEQ ID NO: 85), which isshown in FIG. 39 ; WPRE, which is shown in FIG. 40 ; p108, which isshown in FIG. 41 ; 1OTOF18.CL1, which is shown in FIG. 42 ; 19OTOF48,which is shown in FIG. 43 ; 1OTOF20.CL1, which is shown in FIG. 44 ;21OTOF48.WPRE, which is shown in FIG. 45 ; 1OTOF21.CL1, which is shownin FIG. 46 ; and 22OTOF48.WPRE, which is shown in FIG. 47 ;105.pA.NTF3.CMVd, which is shown in FIG. 48 .

Pairs of these vectors were used to transfect HEK293FT cells. In theseexperiments, 1.2×10⁵ cells/well were seeded in a 24-well plate overnightat 37° C. with 5% CO₂. On the next day, pairs of linearized ITRcontaining plasmids (the pairs shown in FIG. 49 ) were transfected intothe HEK293FT cells using JetPrime reagent (Polyplus). Seventy-two hourspost-transfection, the cells were harvested and lysed using RIPA bufferand analyzed in 4-12% Bolt protein gel. The gel was transferred ontonitrocellulose membrane and blotted with anti-OTOF polyclonal antibody(Thermo PA5-52935). Anti-human beta-actin monoclonal antibody was usedas the primary antibody for internal-loading control between the lanes.Three independent in vitro experiments were performed to analyze andcompare the expression of full-length protein using variousrecombination strategies relative to single plasmid CMV.fl-OTOFtransfection (lane 10).

Compared to the negative control, expression of full-length otoferlinprotein (approximately 226 kD) were observed in every lane except lanenumber 6 (FIGS. 50 and 51 ).

Example 22: In Vivo Recovery of Hearing Function in OTOF^(−/−) MiceUsing Two Vectors

A set of experiments were performed to determine whether hearingfunction could be recovered in Otof^(−/−) mice that have two vectors(p109 and p105; SEQ ID NOs: 84 and 85, respectively) introduced intotheir cochlea. Briefly, a virus preparation (e.g., 1-2×10¹⁰ vg/μL) wasinjected through the auditory bulla covering the round window membrane(RWM) into the scala tympani of the left cochlea at approximatelypostnatal day 6-7 (P6-7). See, e.g., Jung et al. (2015) EMBO J 34:2686-2702, and Al-Moyed et al. (2019) EMBO Molecular Medicine 11(1).pii: e9396. Prior to injection, all mice were anesthetized viaisoflurane and were locally anesthetized, e.g., with xylocainepumpspray, before retroauricular incision.

Auditory brainstem responses (ABRs) were recorded from approximately 3-to 4-week old anesthetized mice subjected to 4, 6, 8, 12, 16, 24, or 32kHz tone burst (e.g., 10 ms plateau, 1 ms cos² rise/fall or 0.03 msbroadband click sound stimuli presented at 20 Hz. See, e.g., Jing et al.(2013) J Neurosci 33: 4456-4467. Ears that were injected were cloggedwith an electrode gel while ABRs were recorded from contralateralnon-injected ears.

ABR click sound thresholds were determined as the lowest sound pressurelevels necessary to evoke reproducible ABR wave response and weremeasured in 10 decibel sound pressure levels (dB SPL) steps from 30 dBSPL to 100 dB SPL. Tone burst thresholds were recorded in 10 dB SPLsteps from 10 dB SPL below the lowest reproducible ABR and up to 90 dBSPL. ABR wave I was defined as the first distinguishable peak betweenthe summating receptor potential (SP) and the prominent ABR wave IIpeak. The amplitude of each ABR wave was calculated as the differencebetween the highest point of a wave and the subsequent local minimum.The summed ABR wave I-V amplitude was calculated by adding up theindividual amplitude values of ABR waves I-V.

The click ABR threshold and the tone burst ABR threshold were determinedin wildtype mice that were not treated, Otof^(−/−) that were nottreated, and Otof^(−/−) mice that were treated with the two vectors(p109 and p105). Hearing function was tested in the treated Otof^(−/−)at days 26-28 and day 91 after administration. The data show thatOtof^(−/−) mice demonstrated significant improvements in hearingfunction after treatment with the two vectors, as compared to untreatedOtof^(−/−) mice (FIGS. 51 and 52 ).

Example 23: In Vivo Recovery of Hearing Function in Human Subjects UsingTwo Vectors

A set of experiments are performed to determine whether hearing functioncould be recovered in human subjects that have two vectors (p109 andp105; SEQ ID NOs: 84 and 85, respectively) introduced into theircochlea. Hearing function is tested in the human subject at days 15, 30,45, 60 and 90 after administration, and is compared to the functionalhearing of human subject that did not receive treatment.

Example 24: In Vitro Expression of Full-Length Human Otoferlin Using TwoVectors

As in other dual vector approaches, two transgenes, each comprising aportion of the full-length transcript, are packaged in separate vectorsand provided, together, to contact a target, e.g., a target cellpopulation in, e.g., a subject in need thereof. The present exampleprovides a set of vectors which were generated to each include a nucleicacid sequence that comprises a portion of the coding sequence of thehuman otoferlin (OTOF) gene or OTOF cDNA. The overall structure andcomponents of the “upstream” AKhOTOF5 vector are shown in FIG. 79 . Thefull 5′ITR-to-3′ITR sequence is represented by SEQ ID NO: 96. Thesequences of the individual components in the order they are found from5′ITR-to-3′ITR are provided in Table 1 below. The overall structure andcomponents of the “downstream” AKhOTOF3 vector are shown in FIG. 80 .The full 5′ITR-to-3′ITR sequence is represented by SEQ ID NO: 105. Thesequences of the individual components in the order they are found from5′ITR-to-3′ITR are provided in Table 2 below. A schematic of the dualAAV vector system is shown in FIG. 78 . An Anc80 capsid (SEQ ID NO: 109;see Table 3 below) independently encapsidates the upstream vector(Anc80.AKhOTOF5) and the downstream vector (Anc80.AKhOTOF3). In someembodiments, when each of the upstream and downstream vectors isadministered to a subject in need thereof, the constructs concatemerizewithin a given cell. In some such embodiments, concatemerizedfull-length OTOF is expressed and generates functional otoferlinprotein.

Pairs of these vectors are used to treat a human subject suffering fromor susceptible to hearing loss. A composition comprising both vectors ofthe dual AAV vector system, Anc80.AKhOTOF5 and Anc80.AKhOTOF3, isintroduced into at least one cochlea of the human subject. Hearingfunction is tested in the human subject at days 15, 30, 45, 60 and 90after administration, and is compared to the functional hearing of thehuman subject prior to receiving treatment or to a human subject thatdid not receive treatment.

AKhOTOF5

The AKhOTOF5 construct comprises two ITRs (SEQ ID NO: 97 and 104), a CAGpromoter (identified by SEQ ID NOs: 98, 99, and 100, comprising a CMVearly enhancer element, chicken beta actin gene sequence, and a chimericintron comprising 3′ splice sequence from the rabbit beta globin gene,respectively), a 5′OTOF coding region (SEQ ID NO: 101), a SD intronsequence (SEQ ID NO: 102), and an AK recombinogenic sequence (SEQ ID NO:103). The full-length AKhOTOF5 is represented by SEQ ID NO: 96.

TABLE 1 AKhOTOF5 SEQ ID Name SEQUENCE NO AKhOTOF5TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCC 96CGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTGTCGACGCGGCCGCACGCGTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAGATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGACCGGTGCCACCATGGCCTTGCTCATCCACCTCAAGACAGTCTCGGAGCTGCGGGGCAGGGGCGACCGGATCGCCAAAGTGACTTTCCGAGGGCAATCCTTCTACTCTCGGGTCCTGGAGAACTGTGAGGATGTGGCTGACTTTGATGAGACATTTCGGTGGCCGGTGGCCAGCAGCATCGACAGAAATGAGATGCTGGAGATTCAGGTTTTCAACTACAGCAAAGTCTTCAGCAACAAGCTCATCGGGACCTTCCGCATGGTGCTGCAGAAGGTGGTAGAGGAGAGCCATGTGGAGGTGACTGACACGCTGATTGATGACAACAATGCTATCATCAAGACCAGCCTGTGCGTGGAGGTCCGGTATCAGGCCACTGACGGCACAGTGGGCTCCTGGGACGATGGGGACTTCCTGGGAGATGAGTCTCTTCAAGAGGAAGAGAAGGACAGCCAAGAGACGGATGGACTGCTCCCAGGCTCCCGGCCCAGCTCCCGGCCCCCAGGAGAGAAGAGCTTCCGGAGAGCCGGGAGGAGCGTGTTCTCCGCCATGAAGCTCGGCAAAAACAGGTCTCACAAGGAGGAGCCCCAAAGACCAGATGAACCGGCGGTGCTGGAGATGGAAGACCTTGACCATCTGGCCATTCGGCTAGGAGATGGACTGGATCCCGACTCGGTGTCTCTAGCCTCAGTCACAGCTCTCACCACTAATGTCTCCAACAAGCGATCTAAGCCAGACATTAAGATGGAGCCAAGTGCTGGGCGGCCCATGGATTACCAGGTCAGCATCACGGTGATCGAGGCCCGGCAGCTGGTGGGCTTGAACATGGACCCTGTGGTGTGCGTGGAGGTGGGTGACGACAAGAAGTACACATCCATGAAGGAGTCCACTAACTGCCCCTATTACAACGAGTACTTCGTCTTCGACTTCCATGTCTCTCCGGATGTCATGTTTGACAAGATCATCAAGATTTCGGTGATTCACTCCAAGAACCTGCTGCGCAGTGGCACCCTGGTGGGCTCCTTCAAAATGGACGTGGGAACCGTGTACTCGCAGCCAGAGCACCAGTTCCATCACAAGTGGGCCATCCTGTCTGACCCCGATGACATCTCCTCGGGGCTGAAGGGCTACGTGAAGTGTGACGTTGCCGTGGTGGGCAAAGGGGACAACATCAAGACGCCCCACAAGGCCAATGAGACCGACGAAGATGACATTGAGGGGAACTTGCTGCTCCCCGAGGGGGTGCCCCCCGAACGCCAGTGGGCCCGGTTCTATGTGAAAATTTACCGAGCAGAGGGGCTGCCCCGTATGAACACAAGCCTCATGGCCAATGTAAAGAAGGCTTTCATCGGTGAAAACAAGGACCTCGTGGACCCCTACGTGCAAGTCTTCTTTGCTGGCCAGAAGGGCAAGACTTCAGTGCAGAAGAGCAGCTATGAGCCCCTGTGGAATGAGCAGGTCGTCTTTACAGACCTCTTCCCCCCACTCTGCAAACGCATGAAGGTGCAGATCCGAGACTCGGACAAGGTCAACGACGTGGCCATCGGCACCCACTTCATTGACCTGCGCAAGATTTCTAATGACGGAGACAAAGGCTTCCTGCCCACACTGGGCCCAGCCTGGGTGAACATGTACGGCTCCACACGTAACTACACGCTGCTGGATGAGCATCAGGACCTGAACGAGGGCCTGGGGGAGGGTGTGTCCTTCCGGGCCCGGCTCCTGCTGGGCCTGGCTGTGGAGATCGTAGACACCTCCAACCCTGAGCTCACCAGCTCCACAGAGGTGCAGGTGGAGCAGGCCACGCCCATCTCGGAGAGCTGTGCAGGTAAAATGGAAGAATTCTTTCTCTTTGGAGCCTTCCTGGAGGCCTCAATGATCGACCGGAGAAACGGAGACAAGCCCATCACCTTTGAGGTCACCATAGGCAACTATGGGAACGAAGTTGATGGCCTGTCCCGAGTAGACCTGATTCAGAACGCAAGTGATGACGAGGCCGGTGATGCCGGGGACCTGGCCTCAGTCTCCTCCACTCCACCAATGCGGCCCCAGGTCACCGACAGGAACTACTTCCATCTGCCCTACCTGGAGCGAAAGCCCTGCATCTACATCAAGAGCTGGTGGCCGGACCAGCGCCGCCGCCTCTACAATGCCAACATCATGGACCACATTGCCGACAAGCTGGAAGAAGGCCTGAACGACATACAGGAGATGATCAAAACGGAGAAGTCCTACCCTGAGCGTCGCCTGCGGGGCGTCCTGGAGGAGCTGAGCTGTGGCTGCTGCCGCTTCCTCTCCCTCGCTGACAAGGACCAGGGCCACTCATCCCGCACCAGGCTTGACCGGGAGCGCCTCAAGTCCTGCATGAGGGAGCTGGAAAACATGGGGCAGCAGGCCAGGATGCTGCGGGCCCAGGTGAAGCGGCACACGGTGCGGGACAAGCTGAGGCTGTGCCAGAACTTCCTGCAGAAGCTGCGCTTCCTGGCGGACGAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATAAGCTTGAATTCAGCTGAGGTGCCTCGGACCGCCTAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGC GCAGAGAGGGAGTGGCCAA 5′ITRTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCC 97CGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCT CMVGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGT 98 enhancerCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTAGATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAGATCTAGGTAT TAGTCATCGCTATTACCATGGCBA gene GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCC 99 sequenceCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCG CGGCGGGCG ChimericGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGCGCCGCC 100 intronTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGC_(TTG)GTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCTGTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGCGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC ATGCCTTCTTCTTTTTCCTACAG5′OTOF ATGGCCTTGCTCATCCACCTCAAGACAGTCTCGGAGCTGCGGGGC 101AGGGGCGACCGGATCGCCAAAGTGACTTTCCGAGGGCAATCCTTCTACTCTCGGGTCCTGGAGAACTGTGAGGATGTGGCTGACTTTGATGAGACATTTCGGTGGCCGGTGGCCAGCAGCATCGACAGAAATGAGATGCTGGAGATTCAGGTTTTCAACTACAGCAAAGTCTTCAGCAACAAGCTCATCGGGACCTTCCGCATGGTGCTGCAGAAGGTGGTAGAGGAGAGCCATGTGGAGGTGACTGACACGCTGATTGATGACAACAATGCTATCATCAAGACCAGCCTGTGCGTGGAGGTCCGGTATCAGGCCACTGACGGCACAGTGGGCTCCTGGGACGATGGGGACTTCCTGGGAGATGAGTCTCTTCAAGAGGAAGAGAAGGACAGCCAAGAGACGGATGGACTGCTCCCAGGCTCCCGGCCCAGCTCCCGGCCCCCAGGAGAGAAGAGCTTCCGGAGAGCCGGGAGGAGCGTGTTCTCCGCCATGAAGCTCGGCAAAAACAGGTCTCACAAGGAGGAGCCCCAAAGACCAGATGAACCGGCGGTGCTGGAGATGGAAGACCTTGACCATCTGGCCATTCGGCTAGGAGATGGACTGGATCCCGACTCGGTGTCTCTAGCCTCAGTCACAGCTCTCACCACTAATGTCTCCAACAAGCGATCTAAGCCAGACATTAAGATGGAGCCAAGTGCTGGGCGGCCCATGGATTACCAGGTCAGCATCACGGTGATCGAGGCCCGGCAGCTGGTGGGCTTGAACATGGACCCTGTGGTGTGCGTGGAGGTGGGTGACGACAAGAAGTACACATCCATGAAGGAGTCCACTAACTGCCCCTATTAGAACGAGTAGTTCGTCTTCGACTTCCATGTCTCTCCGGATGTCATGTTTGACAAGATCATCAAGATTTCGGTGATTCACTCCAAGAACCTGCTGCGCAGTGGCACCCTGGTGGGCTCCTTCAAAATGGACGTGGGAACCGTGTACTCGCAGCCAGAGCACCAGTTCCATCACAAGTGGGCCATCCTGTCTGACCCCGATGACATCTCCTCGGGGCTGAAGGGCTACGTGAAGTGTGACGTTGCCGTGGTGGGCAAAGGGGACAACATCAAGACGCCCCACAAGGCCAATGAGACCGACGAAGATGACATTGAGGGGAACTTGCTGCTCCCCGAGGGGGTGCCCCCCGAACGCCAGTGGGCCCGGTTCTATGTGAAAATTTACCGAGCAGAGGGGCTGCCCCGTATGAACACAAGCCTCATGGCCAATGTAAAGAAGGCTTTCATCGGTGAAAACAAGGACCTCGTGGACCCCTACGTGCAAGTCTTCTTTGCTGGCCAGAAGGGCAAGACTTCAGTGCAGAAGAGCAGCTATGAGCCCCTGTGGAATGAGCAGGTCGTCTTTACAGACCTCTTCCCCCCACTCTGCAAACGCATGAAGGTGCAGATCCGAGACTCGGACAAGGTCAACGACGTGGCCATCGGCACCCACTTCATTGAGCTGCGCAAGATTTCTAATGAGGGAGACAAAGGCTTCCTGCCCACACTGGGCCCAGCCTGGGTGAACATGTACGGCTCCACACGTAACTACACGCTGCTGGATGAGCATCAGGACCTGAACGAGGGCCTGGGGGAGGGTGTGTCCTTCCGGGCCCGGCTCCTGCTGGGCCTGGCTGTGGAGATCGTAGACACCTCCAACCCTGAGCTCACCAGCTCCACAGAGGTGCAGGTGGAGCAGGCCACGCCCATCTCGGAGAGCTGTGCAGGTAAAATGGAAGAATTCTTTCTCTTTGGAGCCTTCCTGGAGGCCTCAATGATCGACCGGAGAAACGGAGACAAGCCCATCACCTTTGAGGTCACCATAGGCAACTATGGGAACGAAGTTGATGGCCTGTCCCGGCCCCAGCGGCCTCGGCCCCGGAAGGAGCCGGGGGATGAGGAAGAAGTAGACCTGATTCAGAACGCAAGTGATGACGAGGCCGGTGATGCCGGGGACCTGGCCTCAGTCTCCTCCACTCCACCAATGCGGCCCCAGGTCACCGACAGGAACTACTTCCATCTGCCCTACCTGGAGCGAAAGCCCTGCATCTACATCAAGAGCTGGTGGCCGGACCAGCGCCGCCGCCTCTACAATGCCAACATCATGGACCACATTGCCGACAAGCTGGAAGAAGGCCTGAACGACATACAGGAGATGATCAAAACGGAGAAGTCCTACCCTGAGCGTCGCCTGCGGGGCGTCCTGGAGGAGCTGAGCTGTGGCTGCTGCCGCTTCCTCTCCCTCGCTGACAAGGACCAGGGCCACTCATCCCGCACCAGGCTTGACCGGGAGCGCCTCAAGTCCTGCATGAGGGAGCTGGAAAACATGGGGCAGCAGGCCAGGATGCTGCGGGCCCAGGTGAAGCGGCACACGGTGCGGGACAAGCTGAGGCTGTGCCAGAACTTCCTGCAGAAGCTGCGCTTCCTGGCGGAC GAG SD intronGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAA 102CTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCT AKGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATT 103TAACAAAAATTTAACGCGAATTTTAACAAAAT 3′ITRAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC 104GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAAAKhOTOF3

The AKhOTOF3 construct comprises two ITRs (SEQ ID NOS: 97 and 104), anAK recombinogenic sequence (SEQ TD NO: 103), an SA intron sequence (SEQID NO: 106), a 3′OTOF coding region (SEQ TD NO: 107), and a bgH polyAsequence (SEQ ID NO: 108). The full-length AKhOTOF3 is represented bySEQ ID NO: 105.

TABLE 2 AKhOTOF3 SEQ ID Name SEQUENCE NO AKhOTOF3TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCC 105CGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTTGTCGACGCGGCCGCACGCGTGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATGATAGGCACCTATTGGTCTTAGTGAGATCCACTTTGCCTTTCTCTCCACAGCCCCAGCACAGCATTCCCGACATCTTCATCTGGATGATGAGCAACAACAAGCGTGTCGCCTATGCCCGTGTGCCCTCCAAGGACCTGCTCTTCTCCATCGTGGAGGAGGAGACTGGCAAGGACTGCGCCAAGGTCAAGACGCTCTTCCTTAAGCTGCCAGGGAAGCGGGGCTTCGGCTCGGCAGGCTGGACAGTGCAGGCCAAGGTGGAGCTGTACCTGTGGCTGGGCCTCAGCAAACAGCGCAAGGAGTTCCTGTGCGGCCTGCCCTGTGGCTTCCAGGAGGTCAAGGCAGCCCAGGGCCTGGGCCTGCATGCCTTCCCACCCGTCAGCCTGGTCTACACCAAGAAGCAGGCGTTCCAGCTCCGAGCGCACATGTACCAGGCCCGCAGCCTCTTTGCCGCCGACAGCAGCGGACTCTCAGACCCCTTTGCCCGCGTCTTCTTCATCAATCAGAGTCAGTGCACAGAGGTGCTGAATGAGACCCTGTGTCCCACCTGGGACCAGATGCTGGTGTTCGACAACCTGGAGCTCTATGGTGAAGCTCATGAGCTGAGGGACGATCCGCCCATCATTGTCATTGAAATCTATGACCAGGATTCCATGGGCAAAGCTGACTTCATGGGCCGGACCTTCGCCAAACCCCTGGTGAAGATGGCAGACGAGGCGTACTGCCCACCCCGCTTCCCACCTCAGCTCGAGTACTACCAGATCTACCGTGGCAACGCCACAGCTGGAGACCTGCTGGCGGCCTTCGAGCTGCTGCAGATTGGACCAGCAGGGAAGGCTGACCTGCCCCCCATCAATGGCCCGGTGGACGTGGACCGAGGTCCCATCATGCCCGTGCCCATGGGCATCCGGCCCGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTACGGGACCTAAAGCGGGTGAACCTGGCCCAGGTGGACCGGCCACGGGTGGACATCGAGTGTGCAGGGAAGGGGGTGCAGTCGTCCCTGATCCACAATTATAAGAAGAACCCCAACTTCAACACCCTCGTCAAGTGGTTTGAAGTGGACCTCCCAGAGAACGAGCTGCTGCACCCGCCCTTGAACATCCGTGTGGTGGACTGCCGGGCCTTCGGTCGCTACACACTGGTGGGCTCCCATGCCGTCAGCTCCCTGCGACGCTTCATCTACCGGCCCCCAGACCGCTCGGCCCCCAGCTGGAACACCACGGTCAGGCTTCTCCGGCGCTGCCGTGTGCTGTGCAATGGGGGCTCCTCCTCTCACTCCACAGGGGAGGTTGTGGTGACTATGGAGCCAGAGGTACCCATCAAGAAACTGGAGACCATGGTGAAGCTGGACGCGACTTCTGAAGCTGTTGTCAAGGTGGATGTGGCTGAGGAGGAGAAGGAGAAGAAGAAGAAGAAGAAGGGCACTGCGGAGGAGCCAGAGGAGGAGGAGCCAGACGAGAGCATGCTGGACTGGTGGTCCAAGTACTTTGCCTCCATTGACACCATGAAGGAGCAACTTCGACAACAAGAGCCCTCTGGAATTGACTTGGAGGAGAAGGAGGAAGTGGACAATACCGAGGGCCTGAAGGGGTCAATGAAGGGCAAGGAGAAGGCAAGGGCTGCCAAAGAGGAGAAGAAGAAGAAAACTCAGAGCTCTGGCTCTGGCCAGGGGTCCGAGGCCCCCGAGAAGAAGAAACCCAAGATTGATGAGCTTAAGGTATACCCCAAAGAGCTGGAGTCCGAGTTTGATAACTTTGAGGACTGGCTGCACACTTTCAACTTGCTTCGGGGCAAGACCGGGGATGATGAGGATGGCTCCACCGAGGAGGAGCGCATTGTGGGACGCTTCAAGGGCTCCCTCTGCGTGTACAAAGTGCCACTCCCAGAGGACGTGTCCCGGGAAGCCGGCTACGACTCCACCTACGGCATGTTCCAGGGCATCCCGAGCAATGACCCCATCAATGTGCTGGTCCGAGTCTATGTGGTCCGGGCCACGGACCTGCACCCTGCTGACATCAACGGCAAAGCTGACCCCTACATCGCCATCCGGCTAGGCAAGACTGACATCCGCGACAAGGAGAACTAGATCTCCAAGCAGCTCAACCCTGTCTTTGGGAAGTCCTTTGACATCGAGGCCTCCTTCCCCATGGAATCCATGCTGACGGTGGCTGTGTATGACTGGGACCTGGTGGGCACTGATGACCTCATTGGGGAAACCAAGATCGACCTGGAGAACCGCTTCTACAGCAAGCACCGCGCCACCTGCGGCATCGCCCAGACCTACTCCACACATGGCTACAATATCTGGCGGGACCCCATGAAGCCCAGCCAGATCCTGACCCGCCTCTGCAAAGACGGCAAAGTGGACGGCCCCCACTTTGGGCCCCCTGGGAGAGTGAAGGTGGCCAACCGCGTCTTCACTGGGCCCTCTGAGATTGAGGACGAGAACGGTCAGAGGAAGCCCACAGACGAGCATGTGGCGCTGTTGGCCCTGAGGCACTGGGAGGACATCCCCCGCGCAGGCTGCCGCCTGGTGCCAGAGCATGTGGAGACGAGGCCGCTGCTCAACCCCGACAAGCCGGGCATCGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACGCCTCTGGACATCTCACCTCGGAAGCCCAAGAAGTACGAGCTGCGGGTCATCATCTGGAACACAGATGAGGTGGTCTTGGAGGACGACGACTTCTTCACAGGGGAGAAGTCCAGTGACATCTTCGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAGCAGGACACAGACGTCCACTACCACTCCCTCACTGGCGAGGGCAACTTCAACTGGCGCTACCTGTTCCCCTTCGACTACCTGGCGGCGGAGGAGAAGATCGTCATCTCCAAGAAGGAGTCCATGTTCTCCTGGGACGAGACCGAGTACAAGATCCCCGCGCGGCTCACCCTGCAGATCTGGGATGCGGACCACTTCTCCGCTGACGACTTCCTGGGGGCCATCGAGCTGGACCTGAACCGGTTCCCGCGGGGCGCAAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTGCCCCTCGTGTCCATCTTCAAGCAAAAGCGCGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAACGATGAGTTTGAGCTCACGGGCAAGGTGGAGGCTGAGCTGCATTTACTGACAGCAGAGGAGGCAGAGAAGAACCCAGTGGGCCTGGCCCGCAATGAACCTGACCCCCTAGAGAAACCCAACCGGCCCGACACGGCCTTCGTCTGGTTCCTCAACCCTCTCAAGTCCATCAAGTACCTCATCTGCACCCGGTACAAGTGGCTCATCATCAAGATCGTGCTGGCGCTGTTGGGGCTGCTCATGTTGGGGCTCTTCCTCTACAGCCTCCCTGGCTACATGGTCAAAAAGCTCCTTGGGGCATGAACTAGTGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGAAGCTTGAATTCAGCTGACGTGCCTCGGACCGCCTAGGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAA 5′ITRTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCC 97CGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACT AGGGGTTCCT AKGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATT 103TAACAAAAATTTAACGCGAATTTTAACAAAAT SA intronGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCT 106 CCACAG 3′OTOFCCCGAGCACAGCATTCCCGAGATCTTCATCTGGATGATGAGCAAC 107AACAAGCGTGTCGCCTATGCCCGTGTGCCCTCCAAGGACCTGCTCTTCTCCATCGTGGAGGAGGAGACTGGCAAGGACTGCGCCAAGGTCAAGACGCTCTTCCTTAAGCTGCCAGGGAAGCGGGGCTTCGGCTCGGCAGGCTGGACAGTGCAGGCCAAGGTGGAGCTGTACCTGTGGCTGGGCCTCAGCAAACAGCGCAAGGAGTTCCTGTGCGGCCTGCCCTGTGGCTTCCAGGAGGTCAAGGCAGCCCAGGGCCTGGGCCTGCATGCCTTCCCACCCGTCAGCCTGGTCTACACCAAGAAGCAGGCGTTCCAGCTCCGAGCGCACATGTACCAGGCCCGCAGCCTCTTTGCCGCCGACAGCAGCGGACTCTCAGACCCCTTTGCCCGCGTCTTCTTCATCAATCAGAGTCAGTGCACAGAGGTGCTGAATGAGACCCTGTGTCCCACCTGGGACCAGATGCTGGTGTTCGACAACCTGGAGCTCTATGGTGAAGCTCATGAGCTGAGGGACGATCCGCCCATCATTGTCATTGAAATCTATGACCAGGATTCCATGGGCAAAGCTGACTTCATGGGCCGGACCTTCGCCAAACCCCTGGTGAAGATGGCAGACGAGGCGTACTGCCCACCCCGCTTCCCACCTCAGCTCGAGTACTACCAGATCTACCGTGGCAACGCCACAGCTGGAGACCTGCTGGCGGCCTTCGAGCTGCTGCAGATTGGACCAGCAGGGAAGGCTGACCTGCCCCCCATCAATGGCCCGGTGGACGTGGACCGAGGTCCCATCATGCCCGTGCCCATGGGCATCCGGCCCGTGCTCAGCAAGTACCGAGTGGAGGTGCTGTTCTGGGGCCTACGGGACCTAAAGCGGGTGAACCTGGCCCAGGTGGACCGGCCACGGGTGGACATCGAGTGTGCAGGGAAGGGGGTGCAGTCGTCCCTGATCCACAATTATAAGAAGAACCCCAACTTCAACACCCTCGTCAAGTGGTTTGAAGTGGACCTCCCAGAGAACGAGCTGCTGCACCCGCCCTTGAACATCCGTGTGGTGGACTGCCGGGCCTTCGGTCGCTACACACTGGTGGGCTCCCATGCCGTCAGCTCCCTGCGACGCTTCATCTACCGGCCCCCAGACCGCTCGGCCCCCAGCTGGAACACCACGGTCAGGCTTCTCCGGCGCTGCCGTGTGCTGTGCAATGGGGGCTCCTCCTCTCACTCCACAGGGGAGGTTGTGGTGACTATGGAGCCAGAGGTACCCATCAAGAAACTGGAGACCATGGTGAAGCTGGACGCGACTTCTGAAGCTGTTGTCAAGGTGGATGTGGCTGAGGAGGAGAAGGAGAAGAAGAAGAAGAAGAAGGGCACTGCGGAGGAGCCAGAGGAGGAGGAGCCAGACGAGAGCATGCTGGACTGGTGGTCCAAGTACTTTGCCTCCATTGACACCATGAAGGAGCAACTTCGACAACAAGAGCCCTCTGGAATTGACTTGGAGGAGAAGGAGGAAGTGGACAATACCGAGGGCCTGAAGGGGTCAATGAAGGGCAAGGAGAAGGCAAGGGCTGCCAAAGAGGAGAAGAAGAAGAAAACTCAGAGCTCTGGCTCTGGCCAGGGGTCCGAGGCCCCCGAGAAGAAGAAACCCAAGATTGATGAGCTTAAGGTATACCCCAAAGAGCTGGAGTCCGAGTTTGATAACTTTGAGGACTGGCTGCACACTTTCAACTTGCTTCGGGGCAAGACCGGGGATGATGAGGATGGCTCCACCGAGGAGGAGCGCATTGTGGGACGCTTCAAGGGCTCCCTCTGCGTGTACAAAGTGCCACTCCCAGAGGACGTGTCCCGGGAAGCCGGCTACGACTCCACCTACGGCATGTTCCAGGGCATCCCGAGCAATGACCCCATCAATGTGCTGGTCCGAGTCTATGTGGTCCGGGCCACGGACCTGCACCCTGCTGACATCAACGGCAAAGCTGACCCCTACATCGCCATCCGGCTAGGCAAGACTGACATCCGCGACAAGGAGAACTACATCTCCAAGCAGCTCAACCCTGTCTTTGGGAAGTCCTTTGACATCGAGGCCTCCTTCCCCATGGAATCCATGCTGACGGTGGCTGTGTATGACTGGGACCTGGTGGGCACTGATGACCTCATTGGGGAAACCAAGATCGACCTGGAGAACCGCTTCTACAGCAAGCACCGCGCCACCTGCGGCATCGCCCAGACCTACTCCACACATGGCTACAATATCTGGCGGGACCCCATGAAGCCCAGCCAGATCCTGACCCGCCTCTGCAAAGACGGCAAAGTGGACGGCCCCCACTTTGGGCCCCCTGGGAGAGTGAAGGTGGCCAACCGCGTCTTCACTGGGCCCTCTGAGATTGAGGACGAGAACGGTCAGAGGAAGCCCACAGACGAGCATGTGGCGCTGTTGGCCCTGAGGCACTGGGAGGACATCCCCCGCGCAGGCTGCCGCCTGGTGCCAGAGCATGTGGAGACGAGGCCGCTGCTCAACCCCGACAAGCCGGGCATCGAGCAGGGCCGCCTGGAGCTGTGGGTGGACATGTTCCCCATGGACATGCCAGCCCCTGGGACGCCTCTGGACATCTCACCTCGGAAGCCCAAGAAGTACGAGCTGCGGGTCATCATCTGGAACACAGATGAGGTGGTCTTGGAGGACGACGACTTCTTCACAGGGGAGAAGTCCAGTGACATCTTCGTGAGGGGGTGGCTGAAGGGCCAGCAGGAGGACAAGCAGGACACAGACGTCCACTAGCACTCCCTCACTGGCGAGGGCAACTTCAACTGGCGCTACCTGTTCCCCTTCGACTACCTGGCGGCGGAGGAGAAGATCGTCATCTCCAAGAAGGAGTCCATGTTCTCCTGGGACGAGACCGAGTACAAGATCCCCGCGCGGCTCACCCTGCAGATCTGGGATGCGGACCACTTCTCCGCTGACGACTTCCTGGGGGCCATCGAGCTGGACCTGAACCGGTTCCCGCGGGGCGCAAAGACAGCCAAGCAGTGCACCATGGAGATGGCCACCGGGGAGGTGGACGTGCCCCTCGTGTCCATCTTCAAGCAAAAGCGCGTCAAAGGCTGGTGGCCCCTCCTGGCCCGCAATGAGAACGATGAGTTTGAGCTCACGGGCAAGGTGGAGGCTGAGCTGCATTTACTGACAGCAGAGGAGGCAGAGAAGAACCCAGTGGGCCTGGCCCGCAATGAACCTGACCCCCTAGAGAAACCCAACCGGCCCGACACGGCCTTCGTCTGGTTCCTCAACCCTCTCAAGTCCATCAAGTACCTCATCTGCACCCGGTACAAGTGGCTCATCATCAAGATCGTGCTGGCGCTGTTGGGGCTGCTCATGTTGGGGCTCTTCCTCTACAGCCTCCCTGGCTACATGGTCAAAAAGCTCCTTGGG GCA bGHpACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCG 108TGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG 3′ITRAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC 104GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGG GAGTGGCCAA

TABLE 3 Anc80L65 SEQ ID Name SEQUENCE NO Anc80L65MAADGYLPDWLEDNLSEGIREWWDL 109 KPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH DKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRV LEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQ TGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNAS GNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFG YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVT TNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQ YGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQA KNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMA THKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATE EYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHT DGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPR PIGTRYLTRNL*

Other Embodiments

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the disclosure in its broader aspects.

While the present disclosure has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the disclosure.

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of thedisclosure, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

What is claimed is:
 1. A plurality of recombinant adeno-associated viral(rAAV) vectors comprising: a) a first rAAV vector comprising a nucleicacid sequence of SEQ ID NO: 96; and b) a second rAAV vector comprising anucleic acid sequence of SEQ ID NO:
 105. 2. The plurality of rAAVvectors of claim 1, wherein the first and second rAAV vectors are eachencapsulated by an AAV capsid.
 3. The plurality of rAAV vectors of claim2, wherein the AAV capsid encapsulating the first rAAV vector is aserotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.
 4. Theplurality of rAAV vectors of claim 2, wherein the AAV capsidencapsulating the second rAAV vector is a serotype selected from any oneof AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8,AAVrh10, AAVrh39, AAVrh43, or Anc80.
 5. The plurality of rAAV vectors ofclaim 2, wherein the first rAAV vector is encapsulated by an Anc80capsid and the second rAAV vector is encapsulated by an Anc80 capsid. 6.The plurality of rAAV vectors of claim 5, wherein the Anc80 capsidscomprise the polypeptide sequence of SEQ ID NO:
 109. 7. A compositioncomprising the plurality of rAAV vectors of claim 1, wherein thecomposition further comprises one or more pharmaceutically acceptablecarriers, diluents, or excipients.
 8. The composition of claim 7,wherein the composition is formulated for intra-cochlear administration.9. The composition of claim 8, wherein the composition is formulated tocomprise a synthetic perilymph solution.
 10. A method of expressing arecombinant full-length otoferlin protein in a mammalian cell, themethod comprising administering a plurality of recombinantadeno-associated viral (rAAV) vectors comprising: a) a first rAAV vectorcomprising a nucleic acid sequence of SEQ ID NO: 96; and b) a secondrAAV vector comprising a nucleic acid sequence of SEQ ID NO: 105; to themammalian cell, wherein the mammalian cell has reduced expression, lackof expression or dysfunction of otoferlin.
 11. The method of claim 10,wherein the mammalian cell is an inner hair cell.
 12. The method ofclaim 10, wherein the mammalian cell is a human cell.
 13. The method ofclaim 10, wherein the mammalian cell comprises a defective otoferlingene.
 14. The method of claim 10, wherein the first and second rAAVvectors are each encapsulated by an AAV capsid.
 15. The method of claim14, wherein the AAV capsid encapsulating the first rAAV vector is aserotype selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, or Anc80.
 16. Themethod of claim 14, wherein the AAV capsid encapsulating the second rAAVvector is a serotype selected from any one of AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, orAnc80.
 17. The method of claim 14, wherein the first rAAV vector isencapsulated by an Anc80 capsid and the second rAAV vector isencapsulated by an Anc80 capsid.
 18. The method of claim 16, wherein theAnc80 capsids comprise the polypeptide sequence of SEQ ID NO:
 109. 19. Amethod of treating hearing loss in a subject having otoferlin relatedhearing loss, the method comprising administering a plurality ofrecombinant adeno-associated viral (rAAV) vectors comprising: a) a firstrAAV vector comprising a nucleic acid sequence of SEQ ID NO: 96; and b)a second rAAV vector comprising a nucleic acid sequence of SEQ ID NO:105; into the cochlea of the subject, wherein the subject has reducedexpression, lack of expression, or dysfunction of otoferlin.
 20. Themethod of claim 19, wherein the subject is a human.
 21. The method ofclaim 19, wherein the first and second rAAV vectors are eachencapsulated by an AAV capsid.
 22. The method of claim 21, wherein theAAV capsid encapsulating the first rAAV vector is a serotype AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39,AAVrh43, or Anc80.
 23. The method of claim 21, wherein the AAV capsidencapsulating the second rAAV vector is a serotype AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43,or Anc80.
 24. The method of claim 21, wherein the first rAAV vector isencapsulated by an Anc80 capsid and the second rAAV vector isencapsulated by an Anc80 capsid.
 25. The method of claim 24, wherein theAnc80 capsids comprise the polypeptide sequence of SEQ ID NO:
 109. 26.The method of claim 19, wherein the plurality of rAAV vectors isadministered as a single dose.
 27. The method of claim 19, wherein theplurality of rAAV vectors is administered as two or more doses.
 28. Themethod of claim 19, wherein the subject has a defective otoferlin gene.